Global amplification using random priming by a composite primer

ABSTRACT

The invention relates to the field of polynucleotide amplification. More particularly, the invention provides methods, compositions and kits for amplification of (i.e., making multiple copies of) a multiplicity of different polynucleotide template sequences using a randomly primed RNA/DNA composite primer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Applications Nos. 60/462,962, filed Apr. 14, 2003,and 60/462,965, filed Apr. 14, 2003, each of which is incorporated byreference in its entirety.

TECHNICAL FIELD

The invention relates to the field of polynucleotide amplification. Moreparticularly, the invention provides methods, compositions and kits foramplifying (i.e., making multiple copies of) a multiplicity of differentpolynucleotide template sequences using a randomly primed RNA/DNAcomposite primer.

BACKGROUND ART

The quality and quantity of nucleic acid (e.g. genomic DNA) sample isimportant for many studies. High-throughput genomic analysis requireslarge amounts of template for testing, yet typically the yield ofnucleic acids from individual patient samples is limited. Forensic andpaleoarcheology work also can be severely limited by nucleic acid samplesize. The limitation of starting material impacts the ability to carryout large scale analysis of multiple parameters, as is required for, forexample, the genotyping of multiple loci in the study of complexdiseases. Moreover, it is well accepted that molecular analysisdetermination of genomic instability in various pathological conditionsuch as cancer, is most precisely carried out in well defined cellpopulations, such as that obtained by laser capture micro-dissection orcell sorting. Nucleic acid amplification technologies that provideglobal amplification of very small polynucleotide samples, for example,from one or a very few cells, may provide a solution to the limitedstarting materials generally available for analysis.

Likewise, the ability to amplify ribonucleic acid (RNA) is an importantaspect of efforts to elucidate biological processes. Total cellular mRNArepresents gene expression activity at a defined time. Gene expressionis affected by cell cycle progression, developmental regulation,response to internal and external stimuli and the like. The profile ofexpressed genes for any cell type in an organism reflects normal ordisease states, response to various stimuli, developmental stages, celldifferentiation, and the like. Non-coding RNAs have been shown to be ofgreat importance in regulation of various cellular functions and incertain disease pathologies. Such RNAs are often present in very lowlevels. Thus, amplification methods capable of amplifying low abundanceRNAs, including RNAs that are not polyadenylated, are of greatimportance.

Various methods for global amplification of DNA target molecules (e.g.,whole genome amplification) have been described, including methods basedon the polymerase chain reaction (PCR). See, e.g., U.S. Pat. Nos.5,731,171; 6,365,375; Daigo et al., (2001) Am. J. Pathol. 158(5):1623-1631; Wang et al, (2001); Cancer Res. 61:4169-4174; Zheng etal, (2001) Cancer Epidemiol. 10:697-700; Dietmaier et al (1999) Am. J.Pathol. 154 (1) 83-95; Stoecklein et al (2002) Am. J. Pathol. 161(1):43-51; U.S. Pat. Nos. 6,124,120; 6,280,949; Dean et al (2002) PNAS99 (8):5261-5266. However, PCR-based global amplification methods, suchas whole genome amplification (WGA), may generate non-specificamplification artifacts, give incomplete coverage of loci, or generateDNA of insufficient length that cannot be used in many applications.PCR-based methods also suffer from the propensity of the PCR reaction togenerate products that are preferentially amplified, and thus resultingin biased representation of genomic sequences in the products of theamplification reaction.

Additionally, a number of methods for the analysis of gene expressionhave been developed in recent years. See, for example, U.S. Pat. Nos.6,251,639, 6,692,918, 6,686,156, 5,744,308; 6,143,495; 5,824,517;5,829,547; 5,888,779; 5,545,522; 5,716,785; 5,409,818; EP 0971039A2;EP0878553A2; and U.S. published patent applications nos. 2002/0115088,2003/0186234, 2003/0087251, and 2004/0023271. These includequantification of specific mRNAs, and the simultaneous quantification ofa large number of mRNAs, as well as the detection and quantification ofpatterns of expression of known and unknown genes. RNA amplification ismost commonly performed using the reverse transcriptase-polymerase chainreaction (RT-PCR) method and variations thereof. These methods are basedon replication of RNA by reverse transcriptase to form single strandedDNA complementary to the RNA (cDNA), which is followed by polymerasechain reaction (PCR) amplification to produce multiple copies of doublestranded DNA. Although these methods are most commonly used, they havesome significant drawbacks: a) the reactions require thermocycling; b)the products are double stranded, thus rendering them less accessible tobinding to probes; and c) the reactions are prone to contamination withproducts of prior amplification, thus requiring strict containment ofreaction mixtures. Other current RNA amplification methods useinitiation of replication of mRNA from the poly-A tail at their 3′ ends.However, not all RNA transcripts have a mRNA tail (for example,prokaryotic RNAs and non-coding RNAs). In addition, due to samplepreparation procedures, the RNA transcript structural integrity iscompromised. Thus, it may be desirable in certain circumstances to useRNA amplification methods that do not require initiation of replicationat the defined poly-A tail. Although analysis of non-amplified RNA isfeasible, a significant amount of starting RNA would be required.However, the total amount of sample RNA that is available is frequentlylimited by the amount of biological sample from which it is derived.Biological samples are often limited in amount and precious. Moreover,the amount of the various RNA species is not equal; some species aremore abundant than others are, and these are more likely and easier, toanalyze. The ability to amplify RNA sequences enables the analysis ofless abundant, rare RNA species. The ability to analyze small samples,by means of nucleic acid amplification, is also advantageous for designparameters of large scale screening of effector molecule libraries, forwhich reduction in sample volume is a major concern both for the abilityto perform very large scale screening or ultra high throughputscreening, and in view of the limiting amounts of library components.

Therefore, there is a need for improved amplification methods,particularly methods which can globally amplify DNA or RNApolynucleotide targets. The invention described herein fulfills thisneed.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

DISCLOSURE OF THE INVENTION

The invention provides methods, compositions, and kits for isothermalglobal amplification using a randomly hybridized RNA/DNA compositeprimer, as well as applications of the amplification methods.

Accordingly, in one aspect, the invention provides methods foramplification of a template polynucleotide, said methods comprising: (a)incubating a reaction mixture, said reaction mixture comprising: (i) atemplate polynucleotide; (ii) a first primer, wherein the first primeris a composite primer that is hybridizable to a multiplicity of templatepolynucleotide sites, wherein the composite primer comprises an RNAportion and a 3′ DNA portion; (iii) a DNA-dependent DNA polymerase; and(iv) an RNA-dependent DNA polymerase (which may be present as a separateenzyme or as an enzyme comprising both DNA-dependent DNA polymerase andRNA-dependent DNA polymerase activities); wherein the incubation isunder conditions that permit composite primer random hybridization,primer extension and, in some embodiments, displacement of the primerextension product from template polynucleotide, whereby a complexcomprising an RNA/DNA partial heteroduplex is generated; and (b)incubating a reaction mixture, said reaction mixture comprising (i) thereaction products generated according to step (a) (or an aliquotthereof); (ii) a composite primer (which may be the same as the firstprimer, or may be a different primer), wherein the composite primercomprises an RNA portion and a 3′ DNA portion; (iii) an DNA-dependentDNA polymerase; and (iv) an agent (such as an enzyme) that cleaves RNAfrom an RNA/DNA hybrid; wherein the incubation is under conditions thatpermit RNA cleavage from an RNA/DNA heteroduplex, primer hybridization,primer extension, and displacement of the primer extension product fromthe complex of (a) when its RNA is cleaved and another composite primerbinds to the template and is extended, whereby multiple copies of apolynucleotide (generally, DNA) amplification product are generated. Inembodiments wherein the template polynucleotide is RNA, the reactionmixture of step (a) further comprises (v) an agent (such as anenzyme)that cleaves RNA from an RNA/DNA hybrid, whereby template RNA iscleaved form the complex comprising template RNA and first primerextension product. In some embodiments, the reaction mixture of step (b)comprises the reaction mixture according to step (a) (or an aliquotthereof). In other embodiments, step (b) is initiated by the addition ofan agent that cleaves RNA from a partial RNA/DNA heteroduplex (such asRNase H), and optionally, a DNA-dependent DNA polymerase, to thereaction mixture of step (a). In some embodiments, the reaction mixtureof step (a) and/or (b) further comprises auxiliary primers. In someembodiments (generally embodiments in which the template polynucleotideis DNA), the RNA-dependent DNA polymerase may be omitted from reactionmixture (a).

In another aspect, the invention provides methods for amplification of atemplate polynucleotide by incubating a reaction mixture, said reactionmixture comprising: (a) a complex comprising a RNA/DNA partialheteroduplex, wherein the complex is generated by incubating a firstreaction mixture, said first reaction mixture comprising: (i) apolynucleotide template; (ii) a first primer; wherein the first primeris a composite primer, the composite primer comprising a RNA portion anda 3′ DNA portion; and wherein the composite primer is capable ofhybridizing to a multiplicity of template polynucleotide sites; (iii) aDNA-dependent DNA polymerase; and (iv) an RNA-dependent DNA polymerase;wherein the incubation is under conditions that permit composite primerrandom hybridization, primer extension and displacement of the primerextension product from template polynucleotide, whereby a complexcomprising an RNA/DNA partial heteroduplex is generated; (b) a compositeprimer, wherein the composite primer comprises an RNA portion and a 3′DNA portion; (c) a DNA-dependent DNA polymerase; and (d) an enzyme thatcleaves RNA from an RNA/DNA hybrid; wherein the incubation is underconditions that permit primer hybridization, primer extension, RNAcleavage from an RNA/DNA heteroduplex, and displacement of compositeprimer from the complex of step (a) when its RNA is cleaved and anothercomposite primer binds and is extended, whereby multiple copies of apolynucleotide amplification product are generated. In some embodiments,the reaction mixture and/or first reaction mixture further comprisesauxiliary primers. In some embodiments wherein the templatepolynucleotide is RNA, template RNA in step (a) is cleaved followingprimer extension via conditions or agents promoting cleavage. In someembodiments, the first reaction mixture further comprises: (v) an agent(such as an enzyme) that cleaves RNA from an RNA/DNA hybrid, wherebytemplate RNA is cleaved from the complex comprising template RNA andcomposite primer extension product. In some embodiments (generally thosein which the template polynucleotide is DNA), the complex comprising aRNA/DNA partial heteroduplex may be generated without the use ofRNA-dependent DNA polymerase.

In another aspect, the invention provides methods for amplification of atemplate polynucleotide by incubating a reaction mixture, said reactionmixture comprising: (a) a complex of a first primer extension productand a second primer extension product, wherein the first primerextension product is generated by extension of a randomly primed firstprimer hybridized to target polynucleotide with a DNA polymerase,wherein the first primer is a composite primer comprising an RNA portionand a 3′ DNA portion, wherein the first primer is capable of hybridizingto a multiplicity of template polynucleotide sites, and wherein thesecond primer extension product is generated by extension of a secondprimer hybridized to the first primer extension product; (b) a compositeprimer that is hybridizable to the second primer extension product,wherein the composite primer comprises an RNA portion and a 3′ DNAportion; (c) a DNA-dependent DNA polymerase; and (d) an enzyme thatcleaves RNA from an RNA/DNA hybrid; wherein the incubation is underconditions that permit primer hybridization, primer extension, RNAcleavage from an RNA/DNA heteroduplex, and displacement of compositeprimer from the complex of step (a) when its RNA is cleaved and anothercomposite primer binds and is extended, whereby multiple copies of apolynucleotide amplification product are generated. In some embodimentswherein the template polynucleotide is RNA, the first primer extensionproduct is generated by extension of a randomly primed first primerhybridized to target RNA with a RNA-dependent DNA polymerase. In someembodiments, the first primer extension product and/or the second primerextension product are generated in the presence of auxiliary primers.

In another aspect, the invention provides methods for amplification of apolynucleotide template comprising: (a) random priming of polynucleotidetemplate strand with a composite primer; wherein the composite primercomprises an RNA portion and a 3′ DNA portion, and wherein the compositeprimer is capable of hybridizing to a multiplicity of templatepolynucleotide sites; and (b) incubating template strand in the presenceof a DNA-dependent DNA polymerase, an RNA-dependent DNA polymerase, andan agent that cleaves RNA from an RNA/DNA, whereby multiple copies ofpolynucleotide amplification product are generated via primer extensionand strand displacement. In some embodiments, random priming occurs inthe presence of a DNA polymerase. In some embodiments, auxiliary primersare included in step (a) and/or step (b). In some embodiments (generallythose in which the polynucleotide template is DNA), the RNA-dependentDNA polymerase is omitted from the incubation.

In another aspect, the invention provides methods for amplification ofRNA template polynucleotides which operate as follows: a multiplicity oftemplate polynucleotide sequences are amplified by incubating a reactionmixture, the reaction mixture comprising: (a) a complex of a firstprimer extension product and a second primer extension product, whereinthe first primer extension product is generated by extension of a firstprimer hybridized to template RNA strand with an RNA-dependent DNApolymerase, wherein the first primer is a composite primer comprising anRNA portion and a 3′ DNA portion, wherein the first primer is capable ofhybridizing to a multiplicity of sites on template RNA; wherein thesecond primer extension product is generated by extension of a secondprimer hybridized to the first primer extension product; and wherein RNAfrom the complex of first and second primer extension product is cleaved(using e.g., an enzyme that cleaves RNA from an RNA/DNA hybrid orconditions permitting cleavage, such as heat and/or alkalineconditions); (b) a composite primer that is hybridizable to the secondprimer extension product, wherein the composite primer comprises an RNAportion and a 3′ DNA portion; (c) a DNA-dependent DNA polymerase; and(d) an enzyme that cleaves RNA from an RNA/DNA hybrid; wherein theincubation is under conditions that permit primer hybridization, primerextension, RNA cleavage from an RNA/DNA heteroduplex, and displacementof composite primer from the complex of step (a) when its RNA is cleavedand another composite primer binds to the second primer extensionproduct and is extended, whereby multiple copies of polynucleotideamplification product are generated. In some embodiments, the complex ofstep (a) is generated in the presence of auxiliary primers. In someembodiments, the second primer comprises fragment(s) of cleaved RNAtemplate.

In another aspect, the invention provides methods for amplification of atemplate polynucleotide by (a) randomly priming a templatepolynucleotide with a first primer, wherein said first primer is acomposite primer that is hybridizable to a multiplicity of templatepolynucleotide sites, wherein the composite primer comprises a RNAportion and a 3′ DNA portion; (b) extending the first primer with a DNApolymerase; (c) cleaving RNA from the first primer with an agent thatcleaves RNA from a RNA/DNA heteroduplex; (d) hybridizing anamplification primer to the template polynucleotide, wherein saidamplification primer is a composite primer comprising a RNA portion anda 3′ DNA portion; (e) extending the hybridized amplification primer bystrand displacement DNA synthesis; and (f) cleaving RNA from theamplification primer with an agent that cleaves RNA from a RNA/DNAheteroduplex, such that another amplification primer can hybridize andbe extended, whereby multiple copies of a polynucleotide amplificationproduct are generated.

In another aspect, the invention provides methods for amplification of atemplate polynucleotide by incubating a reaction mixture including: (a)a polynucleotide template strand; (b) a first primer, wherein said firstprimer is a composite primer comprising a RNA portion and a 3′ DNAportion, and wherein the first primer is capable of hybridizing to amultiplicity of template polynucleotide sites; (c) a DNA-dependent DNApolymerase; (d) a RNA-dependent DNA polymerase; and (e) an agent thatcleaves RNA from a RNA/DNA heteroduplex, whereby multiple copies ofpolynucleotide amplification product are generated by primer extensionand strand displacement. In some embodiments (generally thoseembodiments in which the polynucleotide template is DNA), theRNA-dependent DNA polymerase is omitted from the reaction mixture.

As is clear to one skilled in the art, reference to production of copiesof a polynucleotide (e.g., DNA or RNA) template or copies of apolynucleotide sequence complementary to a polynucleotide templaterefers to products that may contain, comprise or consist of suchsequences. As is evident to one skilled in the art, aspects that referto combining and incubating the resultant mixture also encompassesmethod embodiments which comprise incubating the various mixtures (invarious combinations and/or subcombinations) so that the desiredproducts are formed.

Various embodiments of the composite primer(s) used in the methods ofthe invention are described herein. For example, in some embodiments,the RNA portion of a composite primer is 5′ with respect to the 3′ DNAportion. In still other embodiments, the 5′ RNA portion is adjacent tothe 3′ DNA portion. In other embodiments, the RNA portion of thecomposite primer consists of 7 to about 20 nucleotides and the DNAportion of the composite primer consists of about 5 to about 20nucleotides. In still other embodiments, the RNA portion of thecomposite primer consists of about 10 to about 20 nucleotides and theDNA portion of the composite primer consists of about 7 to about 20nucleotides. In some embodiments the composite primer is selected fromthe following composite primers: 5′-GACGGAUGCGGUCUdCdCdAdGdTdGdT-3 (SEQID NO:1); and 5′-CGUAUUCUGACGACGUACUCdTdCdAdGdCdCdT-3′ (SEQ ID NO:2),wherein italics denote ribonucleotides and “d” denotesdeoxyribonucleotides.

In some embodiments, the composite primer comprises random sequence orpartially randomized sequence, although certain embodiments (such ascertain embodiments wherein the template polynucleotide is RNA) excludethe use of primers comprising random or partially random sequence. Inembodiments utilizing a composite primer with random or partially randomsequence, the composite primer may be a population or pool of differentprimers comprising at least 2, at least 3, at least 4, at least 5, atleast 10, at least 15, at least 20, at least 30, at least 40, at least50, or at least 100 different sequences. In other embodiments, thecomposite primer contains one or more “degenerate” nucleotides that isable to hybridize to multiple different nucleotide bases (e.g., inosine,which is able to hybridize to all four canonical bases).

In some embodiments, the composite primer that hybridizes to targetpolynucleotide (such as mRNA or genomic DNA) and the composite primerused during single primer isothermal amplification (i.e., phase (b) ofthe methods) are the same. In some embodiments, the composite primerthat hybridizes to target polynucleotide (such as mRNA or genomic DNA)and the composite primer used during single primer isothermalamplification are different. In some embodiments, two (or more)different composite primers that hybridize to target polynucleotide areused in the methods of the invention.

The methods are applicable to amplifying any target polynucleotide,including, for example, DNA (such as genomic DNA, including human andother mammalian genomic DNA) and RNA (such as total RNA, mRNA, noncodingRNA and ribosomal RNA). One or more steps may be combined and/orperformed sequentially (often in any order, as long as the requisiteproduct(s) are able to be formed), and, as is evident, the inventionincludes various combinations of the steps described herein. It is alsoevident, and is described herein, that the invention encompasses methodsin which the initial, or first, step is any of the steps describedherein. For example, the methods of the invention do not require thatthe first step be random hybridization of composite primer. Methods ofthe invention encompass embodiments in which later, “downstream” stepsare an initial step.

The enzymes which may be used in the methods and compositions aredescribed herein. For example, the agent (such as an enzyme) thatcleaves RNA may be an RNase H, and the RNA-dependent DNA polymerase maybe reverse transcriptase. The RNA-dependent DNA polymerase may comprisean RNase H enzyme activity, or separate enzymes may be used. Similarly,a DNA polymerase may comprise both RNA-dependent and DNA-dependent DNApolymerase enzyme activities, or separate enzymes may be used. ADNA-dependent DNA polymerase, an RNA-dependent DNA polymerase, and theenzyme that cleaves RNA can also be the same enzyme, or separate enzymescomprising each of these activities may be used.

In some embodiments, methods of the invention are used to generatelabeled polynucleotide products (generally DNA products). In someembodiments of methods for generating labeled DNA products, at least onetype of dNTP used is a labeled dNTP. In other embodiments of methods forgenerating labeled DNA products, a labeled composite primer is used.

The invention also provides methods which employ (usually, analyze) theproducts of the amplification methods of the invention, such asdetection of sequence alteration(s) (e.g., genotyping, nucleic acidmutation detection, analysis of splice variants, and the like);determining presence or absence of a sequence of interest; quantifying asequence of interest; gene expression profiling; subtractivehybridization; preparation of subtractive hybridization probe;differential amplification; preparation of libraries (including genomic,cDNA and differential expression libraries); preparation of animmobilized nucleic acid (which can be a nucleic acid immobilized on amicroarray, preparing labeled probes for analysis on arrays (includinghigh density arrays) for the detection and quantification of sequencesof interest, including, for example, sequence determination, detectingsequence variation and genotyping; comparative genome hybridization;detection and/or identification of novel RNAs; and characterizingnucleic acids using the amplification nucleic acid products generated bythe methods of the invention.

Any of the methods of the invention can be used to generatepolynucleotide products that are suitable for characterization of apolynucleotide sequence of interest in a sample. In one embodiment, theinvention provides methods for characterizing (for example, detecting(presence or absence) and/or quantifying) a polynucleotide sequence ofinterest comprising: (a) amplifying a target polynucleotide by any ofthe methods described herein; and (b) analyzing the amplificationproducts. Step (b) of analyzing the amplification products can beperformed by any method known in the art or described herein, forexample by detecting and/or quantifying amplification products that arehybridized to a probe. These amplification products may or may not belabeled. Any of the methods of the invention can be used to generatepolynucleotide (such as DNA) products that are labeled by incorporatinglabeled nucleotides and/or labeled composite primers into appropriatestep(s) of the methods. These labeled products are particularly suitablefor quantification and/or identification by methods known in the art,which include the use of arrays such as cDNA microarrays andoligonucleotide arrays. In one aspect, the invention provides a methodof characterizing a polynucleotide sequence of interest, comprising (a)amplifying a target polynucleotide by a method described herein togenerate labeled polynucleotide products; and (b) analyzing the labeledpolynucleotide products. In some embodiments, the step of analyzingpolynucleotide products comprises determining amount of said products,whereby the amount of the polynucleotide sequence of interest present ina sample is quantified.

The amplification products can also serve as template for furtheranalysis such as sequence analysis, polymorphism detection (includingmultiplex SNP detection ) using, e.g., oligonucleotide ligation-basedassays, analysis using Invader, Cleavase or limited primer extension,and other methods known in the art. For methods that generally requirelarger volumes of input material, the methods of the invention may beused to “pre” amplify a pool of polynucleotides to generate sufficientinput material for subsequent analysis.

In another embodiment, the polynucleotide products can be analyzed by,for example, contacting them with at least one probe. In someembodiments, the at least one probe is provided as a microarray. Themicroarray can comprise at least one probe immobilized on a solid orsemi-solid substrate fabricated from a material selected from the groupconsisting of paper, glass, ceramics, plastic, polypropylene,polystyrene, nylon, polyacrylamide, nitrocellulose, silicon, othermetals, and optical fiber. A probe can be immobilized on the solid orsemi-solid substrate in a two-dimensional configuration or athree-dimensional configuration comprising pins, rods, fibers, tapes,threads, beads, particles, microtiter wells, capillaries, and cylinders.

In another aspect, the invention provides methods of determining a geneexpression profile in a sample, the methods comprising (a) amplifyingRNA template in a sample using any of the methods described herein; and(b) determining an amount of amplification products of each RNA sequenceof interest in the sample, whereby the gene expression profile of thesample is determined. The invention further provides methods ofdetermining a gene expression profile by determining an amount ofamplification products of each RNA sequence of interest in a sample, thesample comprising multiple copies of RNA template amplified by any ofthe methods described herein, whereby the gene expression profile of thesample is determined.

Additionally, the invention also provides methods for archivingpolynucleotide templates. Because the amplification methods of theinvention provide representative amplification of the sequences of thetemplate polynucleotide, amplified product produced by the instantmethods may be used as an archival source for the original templatepolynucleotide. Accordingly, the invention provides methods forarchiving a polynucleotide template by storing the amplificationproducts produced by the methods of the invention. The archivedamplification products may be analyzed as described herein, or may besubjected to further amplification in accordance with the methods of theinvention.

In another aspect, the invention provides products (e.g., multiplecopies of a template polynucleotide) produced by the methods disclosedherein.

The invention also provides compositions, kits, complexes, reactionmixtures and systems comprising various components (and variouscombinations of the components) used in the amplification methodsdescribed herein.

In another aspect, the invention provides compositions comprising any ofthe complexes (which are generally considered as intermediates withrespect to the final amplification products) described herein.

In another aspect, the invention includes any one or more products(including intermediates) and compositions comprising the products(including intermediates) produced by any aspect of the methods of theinvention.

In another aspect, the invention provides reaction mixtures (orcompositions comprising reaction mixtures) which contain variouscombinations of components described herein.

In another aspect, the invention provides kits for conducting themethods described herein. These kits, in suitable packaging andgenerally (but not necessarily) containing suitable instructions,contain one or more components used in the amplification methods.

In another aspect, the invention provides systems for effecting theamplification methods described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates one embodiment of a composite primer useful in themethods of the present invention. As illustrated in the Figure, thecomposite primer comprises a DNA portion at its 3′ end and an RNAportion at its 5′ end. As discussed herein, it is also possible toemploy a composite primer in which the 3′ DNA portion is followed, inthe direction of its 5′, by an RNA portion, which is followed by aportion which is DNA.

FIG. 2 illustrates a composite primer that hybridizes to multiple siteson a template polynucleotide where differing portions of the compositeprimer are hybridized to template polynucleotide depending on the siteat which it is hybridized.

FIG. 3 illustrates primer extension from composite primers that arehybridized at multiple sites on a template strand, where a compositeprimer extension products is being displaced by primer extension from acomposite primer hybridized at a downstream site on the template strand.

FIG. 4 shows a collection of composite primer extension productscomprising composite primer 1 linked (via extension) to sequencescorresponding to a multiplicity of target polynucleotide sequences.

FIG. 5 shows generation of second primer extension product that israndomly primed by the composite primer.

FIG. 6 shows single primer isothermal amplification using the complexcomprising a RNA/DNA partial heteroduplex as a template for furthercomposite-primer dependent amplification.

FIG. 7 illustrates primer extension from composite primers and auxiliaryprimers that are hybridized at multiple sites on a template strand.

FIG. 8 illustrates generation of a second primer extension productprimed by auxiliary primers hybridized to composite primer extensionproduct.

FIG. 9 shows a photograph of a gel showing amplified reaction productgenerated using a single randomly primed composite primer to amplify amultiplicity of template polynucleotide sequences from human genomicDNA.

MODES FOR CARRYING OUT THE INVENTION

Overview of the Invention and its Advantages

The invention discloses novel methods, compositions and kits for globalamplification. The methods provide for amplification using a compositeprimer that is capable of binding to multiple sites within templatepolynucleotide (e.g., mRNA or genomic DNA), whereby a large multiplicityof template polynucleotide sequences (for example, essentially allgenomic DNA) is amplified. The methods are suitable for use with eitherDNA or RNA template. They generate polynucleotide (generally, DNA)products, which are readily suitable for a variety of uses includingcomparative genome hybridization, expression profiling, and multiplegenotype determinations, e.g., by multiplex analysis by microarraytechnologies. The methods are amenable to automation and do not requirethermal cycling. Thus, one of the major advantages of the methods of theinvention is the ability to amplify an entire pool of sequences (or asubset thereof, depending on the desired extent of amplification), whichis essential for application such as comparative genome hybridization,generation of cDNA libraries, generation of subtractive hybridizationprobes, and array based assays, including multiple genotypedeterminations.

The amplification methods of the invention involve composite primerrandom hybridization, primer extension and displacement of compositeprimer extension product by strand displacement, whereby a complexcomprising a RNA-DNA heteroduplex is generated, followed by compositeprimer-dependent isothermal amplification using the complex as asubstrate for further amplification, an aspect that permits rapidamplification and distinguishes the invention from other stranddisplacement amplification methods, such as MDA.

In one aspect, the methods of the invention involve two phases: (a)composite primer random hybridization to template polynucleotide, primerextension and displacement of composite primer extension product bystrand displacement DNA synthesis, whereby a complex comprising aRNA/DNA partial heteroduplex is generated, and (b) compositeprimer-dependent single primer isothermal amplification using thecomplex comprising a RNA/DNA partial heteroduplex as a substrate forfurther amplification.

The methods generally comprise using specially-designed primers,generally a RNA/DNA composite primer, to randomly prime templatepolynucleotide (such as genomic DNA template, mRNA or noncoding RNA). By“randomly prime” or “random hybridization”, as used herein, it is meantthat the composite primer hybridizes to multiple sites within templatepolynucleotide. In some embodiments, an aspect of the invention isdisplacement of primer extension product from template polynucleotide(s)during primer extension by strand displacement DNA synthesis (e.g., byprimer extension with a DNA polymerase having strand displacementactivity) of primers hybridized at a downstream position(s) on thetemplate.

Thus, the invention provides methods of incubating a reaction mixture,said reaction mixture comprising: (a) a polynucleotide template; (b) acomposite primer; wherein the composite primer comprises an RNA portionand a 3′ DNA portion, and wherein the composite primer is capable ofhybridizing to a multiplicity of template polynucleotide sites; (c) aDNA-dependent DNA polymerase with strand displacement activity; and (d)an RNA-dependent DNA polymerase; wherein the incubation is underconditions that permit composite primer random hybridization, primerextension and displacement of the primer extension product from templatepolynucleotide, whereby a population of intermediate complexes aregenerated that generally includes (a) copies of template polynucleotideand/or copies of the complement of polynucleotide sequence appended (viaextension) to composite primer sequences; and (b) copies of templatepolynucleotide and copies of the complement of template polynucleotideappended (via extension) to the complement of composite primersequences. The intermediate complexes may be double-stranded or may bepartially double stranded. By virtue of the presence of composite primersequences in the intermediate complexes, the complexes comprise aRNA/DNA heteroduplex partial heteroduplex. The RNA portion of theRNA/DNA partial heteroduplex generally is introduced by the RNA portionof the composite primer, and the DNA portion of the heteroduplex is madeof the complement of the RNA portion of the composite primer. Forsimplicity, this population of intermediate complexes is termed “acomplex comprising an RNA/DNA partial heteroduplex.”

Generally, the composite primer comprises at least a 3′ DNA portion thatis capable of randomly priming template polynucleotide. Thus, and as thedescription makes clear, reference to a primer that hybridizes to asequence encompasses embodiments in which at least a portion of theprimer is hybridized, embodiments in which two (or more portions) of theprimer are hybridized (separated by unhybridized (looped out) portionsof the primer), and embodiments in which the entire primer ishybridized.

The complex comprising a RNA/DNA partial heteroduplex is substrate forfurther amplification as follows: an enzyme which cleaves RNA from anRNA/DNA hybrid (such as RNase H) cleaves RNA sequence from the hybrid,leaving a 3′ single stranded DNA sequence available for binding by acomposite primer (which may or may not be the same as the firstcomposite primer). Extension of a bound composite primer byDNA-dependent DNA polymerase produces a primer extension product, whichdisplaces the previously bound cleaved first primer extension product,whereby polynucleotide (generally, DNA) product accumulates. It isunderstood that amplified product generally is a mixture of sense andantisense copies of a given template polynucleotide. For example, if thetemplate polynucleotide is double stranded DNA, the amplificationproduct will correspond to each strand. If the template polynucleotideis single stranded, amplification product will generally be producedthat is the copy of template polynucleotide (sense copy) and thecomplement of the template polynucleotide (antisense copy).

The methods disclosed herein are applicable to the amplification of anytarget polynucleotide, including both DNA (e.g., genomic DNA) and RNA(e.g., mRNA and ribosomal RNA) targets. As is evident from thedescription and shown in the example, the methods of the invention arecomposite-primer dependent. That is, amplification is not observed inthe absence of the composite primer.

In another aspect of the invention, auxiliary primers are present in thereaction mixture comprising template polynucleotide, composite primer,DNA-dependent DNA polymerase and RNA-dependent DNA polymerase. As usedherein, “auxiliary primers” refers to a population of random and/orpartially randomized primers. Inclusion of a population of randomprimers during the amplification is believed to enhance the efficiencyof production of and/or global (template-wide, e.g., providingrepresentative amplification of the template, whether the template isDNA or RNA) coverage of the amplification product.

In certain aspects, global amplification of genomic DNA is exemplifiedherein. It is understood, however, that the amplification methods of theinvention are suitable for amplification of any pool or subset (orpolynucleotides representing a significant proportion of a pool orsubset, depending on desired extent of amplification) ofpolynucleotides.

Accordingly, in one aspect, the invention provides methods foramplification of a template polynucleotide, said methods comprising: (a)incubating a reaction mixture, said reaction mixture comprising: (i) atemplate polynucleotide; (ii) a first primer, wherein the first primeris a composite primer that is hybridizable to a multiplicity of templatepolynucleotide sites, wherein the composite primer comprises an RNAportion and a 3′ DNA portion; (iii) a DNA-dependent DNA polymerase; and(iv) an RNA-dependent DNA polymerase (which may be present as a separateenzyme or as an enzyme comprising both DNA-dependent DNA polymerase andRNA-dependent DNA polymerase activities); wherein the incubation isunder conditions that permit composite primer random hybridization,primer extension and displacement of the primer extension product fromtemplate polynucleotide, whereby a complex comprising an RNA/DNA partialheteroduplex is generated; and (b) incubating a reaction mixture, saidreaction mixture comprising (i) the reaction products generatedaccording to step (a) (or an aliquot thereof); (ii) a composite primer(which may be the same as the first primer, or may be a differentprimer), wherein the composite primer comprises an RNA portion and a 3′DNA portion; (iii) an DNA-dependent DNA polymerase; and (iv) an agent(such as an enzyme) that cleaves RNA from an RNA/DNA hybrid; wherein theincubation is under conditions that permit primer hybridization, primerextension, RNA cleavage from an RNA/DNA heteroduplex, and displacementof the primer extension product (i.e., strand displacement DNAsynthesis) from the complex of (a) when its RNA is cleaved and anothercomposite primer binds to the template and is extended, whereby multiplecopies of a polynucleotide (generally, DNA) amplification product aregenerated. In some embodiments, the reaction mixture of step (b)comprises the reaction mixture according to step (a) (or an aliquotthereof). In other embodiments, step (b) is initiated by the addition ofan agent that cleaves RNA from an RNA/DNA heteroduplex (such as RNaseH), and optionally, a DNA-dependent DNA polymerase, to the reactionmixture of step (a). In some embodiments, the reaction mixture of step(a) and/or (b) further comprises auxiliary primers.

In another aspect, the invention provides methods for amplification of atemplate polynucleotide by incubating a reaction mixture, said reactionmixture comprising: (a) a complex of a first primer extension productand a second primer extension product, wherein the first primerextension product is generated by extension of a randomly primed firstprimer hybridized to target polynucleotide with a DNA polymerase,wherein the first primer is a composite primer comprising an RNA portionand a 3′ DNA portion, wherein the first primer is capable of hybridizingto a multiplicity of template polynucleotide sites, and wherein thesecond primer extension product is generated by extension of a secondprimer hybridized to the first primer extension product; (b) a compositeprimer that is hybridizable to the second primer extension product,wherein the composite primer comprises an RNA portion and a 3′ DNAportion; (c) a DNA-dependent DNA polymerase; and (d) an enzyme thatcleaves RNA from an RNA/DNA hybrid; wherein the incubation is underconditions that permit primer hybridization, primer extension, RNAcleavage from an RNA/DNA heteroduplex, and displacement of compositeprimer from the complex of step (a) when its RNA is cleaved and anothercomposite primer binds and is extended, whereby multiple copies of apolynucleotide amplification product are generated. In some embodiments,the first primer extension product and/or the second primer extensionproduct are generated in the presence of auxiliary primers.

In another aspect of the invention wherein the template polynucleotideis RNA, template RNA is cleaved following random composite primerhybridization and primer extension. Template RNA can be cleaved usingmethods well-known in the art, including cleavage with an agent (such asan enzyme, such as RNase H) that cleaves RNA from an RNA/DNA hybrid,cleavage resulting from heat treatment, and cleavage due to chemicaltreatment (e.g., treatment under alkaline conditions). In someembodiments, the invention provides methods of incubating a reactionmixture, said reaction mixture comprising: (a) an RNA template; (b) acomposite primer; wherein the composite primer comprises an RNA portionand a 3′ DNA portion; and wherein the composite primer is capable ofhybridizing to a multiplicity of sites in template RNA; (c) aDNA-dependent DNA polymerase; (d) an RNA-dependent DNA polymerase; and(e) an enzyme capable of cleaving RNA from an RNA/DNA hybrid; whereinthe incubation is under conditions that permit composite primer randomhybridization, primer extension, template RNA cleavage from an RNA/DNAheteroduplex, whereby a complex comprising an RNA/DNA partialheteroduplex is generated. The complex comprising an RNA/DNA partialheteroduplex is the substrate for further amplification as describedabove (i.e., single primer isothermal amplification). In someembodiments, an auxiliary primer is included in the reaction mixture.

Template polynucleotide may also be prepared from RNA template bysynthesis of cDNA. cDNA synthesis may be accomplished using standardmethods, such as priming with random primers (e.g., random hexamerdeoxyoligonucleotides) and primer extension with a RNA-dependent DNApolymerase (e.g., reverse transcripatase) in the presence of dNTPs andappropriate reaction conditions (e.g., temperature, pH and ionicconditions). Only the first strand cDNA synthesis need be performed, asfirst strand cDNA synthesis will produce a DNA polynucleotide that canbe amplified in accordance with the methods of the invention.

The methods of the invention include methods using the amplificationproducts (so-called “applications”). The invention also provides methodswhich employ (usually, analyze) the products of the amplificationmethods of the invention, such as methods of nucleic acid mutationdetection (including methods of genotyping), determining the presence orabsence of a sequence of interest, quantitating a sequence of interest,preparation of an immobilized nucleic acid, comparative genomichybridization, discovery of novel nucleic acid sequences, andcharacterizing nucleic acids using the amplified nucleic acid productsgenerated by the methods of the invention.

Any of the methods of the invention can be used to generatepolynucleotide products that are suitable for characterization of apolynucleotide sequence of interest in a sample. In one embodiment, theinvention provides methods for characterizing (for example, detecting(presence or absence) and/or quantifying) a polynucleotide sequence ofinterest comprising: (a) amplifying a target polynucleotide by any ofthe methods described herein; and (b) analyzing the amplificationproducts. Step (b) of analyzing the amplification products can beperformed by any method known in the art or described herein, forexample by detecting and/or quantifying amplification products that arehybridized to a probe. These amplification products may or may not belabeled. Any of the methods of the invention can be used to generatepolynucleotide (such as DNA) products that are labeled by incorporatinglabeled nucleotides and/or labeled composite primers into appropriatestep(s) of the methods. These labeled products are particularly suitablefor quantification and/or identification by methods known in the art,which include the use of arrays such as cDNA microarrays andoligonucleotide arrays.

The invention provides methods to characterize (for example, detectpresence or absence of and/or quantify) an polynucleotide sequence ofinterest by generating polynucleotide products using amplificationmethods of the invention, and analyzing the products bydetection/quantification methods such as those based on arraytechnologies or solution phase technologies. These amplificationproducts may be labeled or unlabeled.

The methods of the invention may be used to amplify a pool ofpolynucleotides (or polynucleotides representing a significantproportion of a pool, depending on desired extent of amplification) togenerate sufficient input material for subsequent analysis. Thus, and asis described herein, amplification products can also serve as templatefor further analysis such as sequence, polymorphism detection (includingmultiplex SNP detection) using, e.g., oligonucleotide ligation-basedassays, analysis using Invader, Cleavase or limited primer extension,and the like. Amplification product may also serve as template forfurther amplification by the methods of the invention or otheramplification method known in the art.

In yet another embodiment, the invention provides methods forimmobilizing nucleic acids, including methods for generating microarraysof nucleic acids, using amplification products of the amplificationmethods of the invention.

In another embodiment, the invention provides methods of generating cDNAlibraries, methods of generating subtractive hybridization probes, andmethods of generating subtractive hybridization libraries.

Advantages of the Invention

Various methods for global amplification of nucleic acids have beendeveloped. PCR-based methods, such as PEP, may generate non-specificamplification artifacts, give incomplete coverage of loci, or generateDNA product of insufficient length that cannot be used in manyapplications. PCR-based methods also suffer from the propensity of thePCR reaction to generate products that are preferentially amplified, andthus resulting in biased representation of genomic sequences in theproducts of the amplification reaction. Also, PCR-based methods requirecumbersome thermal cycling.

Additionally, a number of methods for the detection and quantificationof gene expression levels have been developed in recent years. Forexample, microarrays, in which either defined cDNAs or oligonucleotidesare immobilized at discrete locations on, for example, solid orsemi-solid substrates, or on defined particles, enable the detectionand/or quantification of the expression of a multitude of genes in agiven specimen. Using these previously known methods to detect presenceof absence of and/or quantify multiple RNA species in a sample, which inturn is used as a measure of gene expression profiling, generallyrequires direct labeling of cDNA, which requires a large amount of inputtotal RNA. Thus, when using total RNA preparations from a given cell ortissue sample to analyze mRNA, the analysis of gene expression in thesample using methods such as arrays requires a substantial amount ofinput RNA, which generally ranges from 50 to 200 μg. Similarly, 2 to 5μg of mRNA purified from a total RNA preparation would generally berequired for a single analysis of gene expression profiling using arraytechnologies. This is a clear limitation of methods such as those basedon array technology, insofar as the number of cells, or size of tissuespecimen required is very large, and these cells or tissue specimens areoften scarcely available for testing or are too precious. Thislimitation is especially severe in the study of clinical specimens,where the cells to be studied are rare and/or difficult to cultivate invitro, and in high throughput screening of libraries of effectormolecules. Also, previous transcription-based methods of amplificationof mRNA (described in, for example, Lockhart et al, Nature Biotechnology(1996), 14, 1675-1680); van Gelder et al., U.S. Pat. No. 5,716,785), arelimited to the amplification efficiency of DNA-dependent RNA polymerasesand some of these methods require multiple steps. Moreover, the processby which the polymerase promoter sequence is incorporated is prone toresult in non-specific amplification.

The methods of the invention offer the ability to efficiently amplifytemplate polynucleotides (including both RNA and DNA). Thus, the utilityof detection/quantification methods which ca be used with theamplification products of the invention, such as those based on arraytechnology, real time PCR, quantitative TaqMan, quantitative PCR usingmolecular beacons, and the like, should be greatly enhanced.

The methods of the invention do not require thermocycling and all of thesteps can be performed isothermally, although the various steps may becarried out a different temperatures. This feature provides numerousadvantages, including facilitating automation and adaptation for highthrough-put procedures. The isothermal reaction is generally faster thanthat afforded by thermal cycling, and is suitable for performing themethods of the invention in miniaturized devices.

The ability to efficiently amplify pools of template polynucleotidesequences (such as genomic DNA) under conditions that will generally notalter the representation of the nucleic acid sequences in the startingsample, will greatly enhance the utility of the detection/quantificationmethods such as those based on the powerful array technology.

The ability to efficiently amplify RNA using the random initiation ofreplication according the methods of the invention provides means forrepresenting all sequences in the pool of sequences (or representing asignificant proportion of a pool depending on desired extent of theamplification) in the sample, for example, sequences representing thefull length of mRNA species in a sample. The methods of the invention donot rely on oligo-dT primers (to bind the poly-A tail of mRNA) toinitiate amplification; thus, the methods may be used to amplifynon-poly-A tailed RNAs, such as noncoding RNAs and RNAs ofnon-eukaryotic species. The methods of the invention do not requireprior knowledge of the sequences in the sample and are thus suitable fordiscovery of novel transcripts, even when present in low abundanceand/or representing non-coding transcripts. The ability to efficientlyamplify RNA under conditions that will generally not alter therepresentation of the nucleic acid sequences in the preparation, willgreatly enhance the utility of the detection/quantification methods suchas those based on the powerful array technology.

General Techniques

The practice of the invention will employ, unless otherwise indicated,conventional techniques of molecular biology (including recombinanttechniques), microbiology, cell biology, biochemistry, and immunology,which are within the skill of the art. Such techniques are explainedfully in the literature, such as, “Molecular Cloning: A LaboratoryManual”, second edition (Sam brook et al., 1989); “OligonucleotideSynthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I.Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.);“Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds.,1987, and periodic updates); “PCR: The Polymerase Chain Reaction”,(Mullis et al., eds., 1994).

Primers, oligonucleotides and polynucleotides employed in the inventioncan be generated using standard techniques known in the art.

Definitions

A “template,” “template strand,” “template polynucleotide,” “templateDNA,” target sequence,” “target nucleic acid,” or “target DNA,” “targetpolynucleotide,” “template RNA,” or “target RNA,” as used herein, is apolynucleotide for which amplification is desired. The templatepolynucleotide can comprise DNA or RNA. The template sequence may beknown or not known, in terms of its actual sequence. Generally, theterms “target,” “template,” and variations thereof, are usedinterchangeably.

“Amplification,” or “amplifying”, as used herein, generally refers tothe process of producing multiple copies of a desired sequence.“Multiple copies” mean at least 2 copies. A “copy” does not necessarilymean perfect sequence complementarity or identity to the templatesequence. For example, copies can include nucleotide analogs such asdeoxyinosine, intentional sequence alterations (such as sequencealterations introduced through a primer comprising a sequence that ishybridizable, but not complementary, to the template, or a non-targetsequence introduced through a primer), and/or sequence errors that occurduring amplification. “Amplifying” a sequence may generally refer tomaking copies of a sequence or its complement, with the understandingthat, as stated above, copying does not necessarily mean perfectsequence complementarity or identity with respect to the templatesequence.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase. A polynucleotidemay comprise modified nucleotides, such as methylated nucleotides andtheir analogs. If present, modification to the nucleotide structure maybe imparted before or after assembly of the polymer. The sequence ofnucleotides may be interrupted by non-nucleotide components. Apolynucleotide may be further modified after polymerization, such as byconjugation with a labeling component. Other types of modificationsinclude, for example, “caps”, substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, poly-L-lysine, etc.), those with intercalators (e.g.,acridine, psoralen, etc.), those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.), those containingalkylators, those with modified linkages (e.g., alpha anomeric nucleicacids, etc.), as well as unmodified forms of the polynucleotide(s).Further, any of the hydroxyl groups ordinarily present in the sugars maybe replaced, for example, by phosphonate groups, phosphate groups,protected by standard protecting groups, or activated to prepareadditional linkages to additional nucleotides, or may be conjugated tosolid supports. The 5′ and 3′ terminal OH can be phosphorylated orsubstituted with amines or organic capping groups moieties of from 1 to20 carbon atoms. Other hydroxyls may also be derivatized to standardprotecting groups. Polynucleotides can also contain analogous forms ofribose or deoxyribose sugars that are generally known in the art,including, for example, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or2′-azido-ribose, carbocyclic sugar analogs, a-anomeric sugars, epimericsugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanosesugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogssuch as methyl riboside. One or more phosphodiester linkages may bereplaced by alternative linking groups. These alternative linking groupsinclude, but are not limited to, embodiments wherein phosphate isreplaced by P(O)S(“thioate”), P(S)S (“dithioate”), “(O)NR₂ (“amidate”),P(O)R, P(O)OR′, CO or CH₂ (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

A “labeled dNTP,” or “labeled rNTP,” as used herein, refers,respectively, to a dNTP or rNTP, or analogs thereof, that is directly orindirectly attached with a label. For example, a “labeled” dNTP or rNTP,may be directly labeled with, for example, a dye and/or a detectablemoiety, such as a member of a specific binding pair (such asbiotin-avidin). A “labeled” dNTP or rNTP, may also be indirectly labeledby its attachment to, for example, a moiety to which a label is/can beattached. A dNTP or rNTP, may comprise a moiety (for example, an aminegroup) to which a label may be attached following incorporation of thedNTP or rNTP into an extension product. Useful labels in the presentinvention include fluorescent dyes (e.g., fluorescein isothiocyanate,Texas red, rhodamine, green fluorescent protein and the like),radioisotopes (e.g., ³H, ³⁵S, ³²P, ³³P, ¹²⁵I, or ¹⁴C), enzymes (e.g.,LacZ, horseradish peroxidase, alkaline phosphatase, ), digoxigenin, andcolorimetric labels such as colloidal gold or colored glass or plastic(e.g., polystyrene, polypropylene, latex, etc.) beads. Variousanti-ligands and ligands can be used (as labels themselves or as a meansfor attaching a label). In the case of a ligand that has a naturalanti-ligand, such as biotin, thyroxine and cortisol, the ligand can beused in conjunction with labeled anti-ligands.

The “type” of dNTP or rNTP, as used herein, refers to the particularbase of a nucleotide, namely adenine, cytosine, thymine, uridine, orguanine.

“Oligonucleotide,” as used herein, generally refers to short, generallysingle stranded, generally synthetic polynucleotides that are generally,but not necessarily, less than about 200 nucleotides in length.Oligonucleotides in the invention include the composite primer andauxiliary primer. The terms “oligonucleotide” and “polynucleotide” arenot mutually exclusive. The description above for polynucleotides isequally and fully applicable to oligonucleotides.

A “primer,” as used herein, refers to a nucleotide sequence, generallywith a free 3′-OH group, that hybridizes with a template sequence (suchas a target polynucleotide, target DNA, or a primer extension product)and is capable of promoting polymerization of a polynucleotidecomplementary to the template. A “primer” can be, for example, anoligonucleotide.

“Auxiliary primers” as used herein, are a population of primerscomprising randomized and/or partially-randomized sequences. Auxiliaryprimers are a polynucleotide as described herein, though generally,auxiliary primers are made of DNA. Random primers are known in the artand are commercially available. An example of auxiliary primers is thepopulation of randomized hexamer primers shown in Example 1.

To “inhibit” is to decrease or reduce an activity, function, and/oramount as compared to a reference.

By “randomly prime” or “random hybridization”, as used herein, it ismeant that the composite primer hybridizes to multiple sites within thetemplate polynucleotide.

As used herein, “complex comprising an RNA/DNA partial heteroduplex”generally refers to a population of intermediate complexes thatgenerally includes (a) copies of template polynucleotide and/or copiesof the complement of template polynucleotide sequence appended tocomposite primer sequences; and (b) copies of template polynucleotideand/or copies of the complement of template polynucleotide appended tothe complement of composite primer sequences. By virtue of the presenceof composite primer sequence in the intermediate complexes, thecomplexes comprise at least a RNA/DNA partial heteroduplex. The RNAportion of the partial heteroduplex generally is introduced (viaextension) by the RNA portion of the composite primer, and the DNAportion of the partial heteroduplex comprises the complement of the RNAportion of the composite primer. As discussed herein, the complexcomprising an RNA/DNA partial heteroduplex functions as a substrate forfurther amplification during the single primer isothermal amplificationphase of the methods. Generally, RNA in the RNA/DNA partial heteroduplexis cleaved, generating a 3′ single stranded portion with sequences thatare complementary to RNA in a composite primer (and thus forming abinding site for a composite primer). Thus, reference to “a complexcomprising a 3′ single stranded portion” generally refers to the complexcomprising an RNA/DNA partial heteroduplex when its RNA is cleaved.

A “complex” is an assembly of components. A complex may or may not bestable and may be directly or indirectly detected. For example, as isdescribed herein, given certain components of a reaction, and the typeof product(s) of the reaction, existence of a complex can be inferred.For purposes of this invention, a complex is generally an intermediatewith respect to the final amplification product(s). An example of acomplex is a complex of composite primer extension product and secondcomposite primer extension product, as described herein.

A “portion” or “region,” used interchangeably herein, of apolynucleotide or oligonucleotide is a contiguous sequence of 2 or morebases. In other embodiments, a region or portion is at least about anyof 3, 5, 10, 15, 20, 25 or more contiguous nucleotides.

A region, portion, or sequence which is “adjacent” to another sequencedirectly abuts that region, portion, or sequence. For example, an RNAportion which is adjacent to a 5′ DNA portion of a composite primerdirectly abuts that region.

A “reaction mixture” is an assemblage of components, which, undersuitable conditions, react to form a complex (which may be anintermediate) and/or a product(s).

“A”, “an” and “the”, and the like, unless otherwise indicated includeplural forms. “A” fragment means one or more fragments.

Conditions that “allow” an event to occur or conditions that are“suitable” for an event to occur, such as hybridization, strandextension, and the like, or “suitable” conditions are conditions that donot prevent such events from occurring. Thus, these conditions permit,enhance, facilitate, and/or are conducive to the event. Such conditions,known in the art and described herein, depend upon, for example, thenature of the nucleotide sequence, temperature, and buffer conditions.These conditions also depend on what event is desired, such ashybridization, cleavage, and/or strand extension.

Sequence “mutation,” as used herein, refers to any sequence alterationin a sequence of interest in comparison to a reference sequence. Areference sequence can be a wild type sequence or a sequence to whichone wishes to compare a sequence of interest. A sequence mutationincludes single nucleotide changes, or alterations of more than onenucleotide in a sequence, due to mechanisms such as substitution,transversion, deletion or insertion. Single nucleotide polymorphism(SNP) is also a sequence mutation as used herein.

“Microarray” and “array,” as used interchangeably herein, comprise asurface with an array, preferably ordered array, of putative binding(e.g., by hybridization) sites for a biochemical sample (target) whichoften has undetermined characteristics. In a preferred embodiment, amicroarray refers to an assembly of distinct polynucleotide oroligonucleotide probes immobilized at defined locations on a substrate.Arrays are formed on substrates fabricated with materials such as paper,glass, plastic (e.g., polypropylene, nylon, polystyrene),polyacrylamide, nitrocellulose, silicon, optical fiber or any othersuitable solid or semi-solid support, and configured in a planar (e.g.,glass plates, silicon chips) or three-dimensional (e.g., pins, fibers,beads, particles, microtiter wells, capillaries) configuration. Probesforming the arrays may be attached to the substrate by any number ofways including (i) in situ synthesis (e.g., high-density oligonucleotidearrays) using photolithographic techniques (see, Fodor et al., Science(1991), 251:767-773; Pease et al., Proc. Natl. Acad. Sci. U.S.A. (1994),91:5022-5026; Lockhart et al., Nature Biotechnology (1996), 14:1675;U.S. Pat. Nos. 5,578,832; 5,556,752; and 5,510,270); (ii)spotting/printing at medium to low-density (e.g., cDNA probes) on glass,nylon or nitrocellulose (Schena et al, Science (1995), 270:467-470,DeRisi et al, Nature Genetics (1996), 14:457-460; Shalon et al., GenomeRes. (1996), 6:639-645; and Schena et al., Proc. Natl. Acad. Sci. U.S.A.(1995), 93:10539-11286); (iii) by masking (Maskos and Southern, Nuc.Acids. Res. (1992), 20:1679-1684) and (iv) by dot-blotting on a nylon ornitrocellulose hybridization membrane (see, e.g., Sambrook et al., Eds.,1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Vol. 1-3, ColdSpring Harbor Laboratory (Cold Spring Harbor, N.Y.)). Probes may also benoncovalently immobilized on the substrate by hybridization to anchors,by means of magnetic beads, or in a fluid phase such as in microtiterwells or capillaries. The probe molecules are generally nucleic acidssuch as DNA, RNA, PNA, and cDNA but may also include proteins,polypeptides, oligosaccharides, cells, tissues and any permutationsthereof which can specifically bind the target molecules.

The term “3′” generally refers to a region or position in apolynucleotide or oligonucleotide 3′ (downstream) from another region orposition in the same polynucleotide or oligonucleotide.

The term “5′” generally refers to a region or position in apolynucleotide or oligonucleotide 5′ (upstream) from another region orposition in the same polynucleotide or oligonucleotide.

The term “3′-DNA portion,” “3′-DNA region,” “3′-RNA portion,” and“3′-RNA region,” refer to the portion or region of a polynucleotide oroligonucleotide located towards the 3′ end of the polynucleotide oroligonucleotide, and may or may not include the 3′ most nucleotide(s) ormoieties attached to the 3′ most nucleotide of the same polynucleotideor oligonucleotide. The 3′ most nucleotide(s) can be preferably fromabout 1 to about 50, more preferably from about 10 to about 40, evenmore preferably from about 20 to about 30 nucleotides.

The term “5′-DNA portion,” “5′-DNA region,” “5′-RNA portion,” and“5′-RNA region,” refer to the portion or region of a polynucleotide oroligonucleotide located towards the 5′ end of the polynucleotide oroligonucleotide, and may or may not include the 5′ most nucleotide(s) ormoieties attached to the 5′ most nucleotide of the same polynucleotideor oligonucleotide. The 5′ most nucleotide(s) can be preferably fromabout 1 to about 50, more preferably from about 10 to about 40, evenmore preferably from about 20 to about 30 nucleotides.

“Absent” or “absence” of product, and “lack of detection of product” asused herein includes insignificant, or de minimus levels, generally dueto lack of significant accumulation of product.

In accordance with well established principles of patent law“comprising” means “including.”

Amplification Methods of the Invention

The following are examples of the amplification methods of theinvention. It is understood that various other embodiments may bepracticed, given the general description provided above. For example,reference to using a composite primer means that any of the compositeprimers described herein may be used.

Amplification Using a Composite Primer that Hybridizes to a Multiplicityof Template Polynucleotide Sites

The invention provides methods for global amplification using acomposite primer that is capable of binding to multiple sites withintemplate polynucleotide, including DNA and RNA template polynucleotides.

Generally, the methods of the invention involve two phases. (a)composite primer random hybridization, primer extension and displacementof composite primer extension product by strand displacement, whereby acomplex comprising a RNA/DNA partial heteroduplex is generated, and (b)composite-primer dependent single primer isothermal amplification.

Thus, the methods of the invention work as follows: (a) incubating areaction mixture, said reaction mixture comprising: (i) a templatepolynucleotide; (ii) a first primer, wherein the first primer is acomposite primer that is hybridizable to a multiplicity of templatepolynucleotide sites, wherein the composite primer comprises an RNAportion and a 3′ DNA portion; (iii) a DNA-dependent DNA polymerase; and(iv) an RNA-dependent DNA polymerase (which may be present as a separateenzyme or as an enzyme comprising both DNA-dependent DNA polymerase andRNA-dependent DNA polymerase activities); wherein the incubation isunder conditions that permit composite primer random hybridization,primer extension and, in some embodiments, displacement of the primerextension product from template polynucleotide, whereby a complexcomprising an RNA/DNA partial heteroduplex is generated; and (b)incubating a reaction mixture, said reaction mixture comprising (i) thereaction products generated according to step (a) (or an aliquotthereof); (ii) a composite primer (which may be the same as the firstprimer or may different from the first primer); (iii) an DNA-dependentDNA polymerase; and (iv) an agent (such as an enzyme) that cleaves RNAfrom an RNA/DNA hybrid; wherein the incubation is under conditions thatpermit primer hybridization, primer extension, RNA cleavage from anRNA/DNA heteroduplex, and displacement of the primer extension productwhen its RNA is cleaved and another composite primer binds to thetemplate and is extended, whereby multiple copies of a polynucleotide(generally, DNA) amplification product are generated. In someembodiments, the reaction mixture of step (b) comprises the reactionmixture according to step (a) (or an aliquot thereof). In otherembodiments, step (b) is initiated by the addition of an agent thatcleaves RNA from an RNA/DNA heteroduplex (such as RNase H) to thereaction mixture of step (a). In embodiments in which the templatepolynucleotide is RNA, the reaction mixture of step (a) furthercomprises an agent (such as an enzyme) that that cleaves RNA from anRNA/DNA heteroduplex.

(a) Composite Primer Random Hybridization, Primer Extension andDisplacement of Composite Primer Extension Product by StrandDisplacement

The methods generally comprise using specially-designed primers,generally a RNA/DNA composite primer. In a first phase of theamplification methods, composite primer is used to randomly primetemplate polynucleotide (such as genomic DNA). By “randomly prime” or“random hybridization”, as used herein, it is meant that the compositeprimer hybridizes to multiple sites within template polynucleotide. Wehave discovered that certain composite primers bind a multiplicity ofsites within template polynucleotide (generally, under conditionspromoting random primer hybridization) and are thus particularlysuitable for use in the methods of the invention. Generally, suitablecomposite primers show partial homology to a multiplicity of templatenucleic acid sequences, particularly in the 3′ sequences of thecomposite primer, and thus, the composite primer comprises at least a 3′DNA portion that is capable of randomly priming template polynucleotide(particularly under conditions permitting random primer hybridization).Selection and design of composite primers is described further below.

Various embodiments of the composite primer and used in the methods ofthe invention are described herein. For example, FIG. 1 illustrates oneembodiment of a composite primer useful in the methods of the presentinvention. As illustrated in the Figure, the composite primer comprisesa DNA portion at its 3′ end and an RNA portion at its 5′ end. Asdiscussed herein, it is also possible to employ a composite primer inwhich the 3′ DNA portion is followed, in the direction of its 5′, by anRNA portion, which is followed by a portion which is DNA. The length ofeach of these sections is generally determined for maximum efficiency ofthe amplification. In some embodiments, the composite primer thathybridizes to target polynucleotide and the composite primer used duringsingle primer isothermal amplification are the same. In someembodiments, the composite primer that hybridizes to targetpolynucleotide and the composite primer used during single primerisothermal amplification are different.

Reference to a primer that binds (hybridizes to) a sequence (ortemplate) encompasses embodiments in which at least a portion of theprimer is hybridized, as well as those embodiments in which an entireprimer is hybridized. Thus, and as the description makes clear,reference to a primer that hybridizes to a sequence encompassesembodiments in which at least a portion of the primer is hybridized aswell as embodiments in which two (or more portions) of the primer arehybridized, separated by unhybridized (looped out) portions of theprimer, and embodiments in which the entire primer is hybridized. Forexample, FIG. 2 illustrates a single composite primer that hybridizes tomultiple positions on a template polynucleotide where differing portionsof the composite primer are hybridized to template polynucleotidedepending on the site (sequence) at which it is hybridized. Thus,according to the methods of the invention, only a portion of the 3′-endof the composite primer must be hybridized in order for initiation ofprimer extension by DNA polymerase. In some embodiments, for example,only 2, 3, 4, 5, 6, 7, 8 or more nucleotides of the 3′ end of the primerneed to hybridize in order for primer extension to be initiated. It isunderstood that hybridization of the 3′-most portion of the compositeprimer may be stabilized to various extents by further hybridization ofanother portion of the primer (with or without looping out ofintervening primer portions). A DNA polymerase can be included duringprimer hybridization to enhance (e.g., stabilize) hybridization ofcomposite primer by initiation of primer extension, and thus,stabilization of primer hybridization.

Random hybridization of the composite primer to template polynucleotidegenerally occurs under conditions permitting random (nonspecific) primerhybridization. Such conditions are well known in the art and include:decreased stringency during primer hybridization and/or primer extension(including reduced temperature and/or buffer conditions of reducedstringency); composite primer selection and/or design (discussed furtherherein); and composite primer and template concentration. It isunderstood that stringency of hybridization, composite primer selectionand/or primer concentration may be used to control the frequency ofcomposite primer hybridization, and thus to control coverage and/orrepresentation of template sequences in amplification product. As notedabove, an aspect of the invention is displacement of intervening primerextension product during primer extension of composite primershybridized at downstream position(s) on the template, whereby primerextension products are displaced from template polynucleotide.Preferably, a DNA polymerase is used that possesses stand displacementactivity.

Composite primer random hybridization, primer extension and displacementof primer extension product by strand displacement results in generationof a multiplicity of complexes comprising a RNA/DNA partialheteroduplex. The complexes comprise (a) copies of templatepolynucleotide and/or copies of the complement of polynucleotidesequence appended (via extension) to composite primer sequences; and (b)copies of template polynucleotide and copies of the complement oftemplate polynucleotide appended (via extension) to the complement ofcomposite primer sequences. Generally, the RNA portion of the complex isintroduced by the composite primer.

In some embodiments, generation of complexes comprising a RNA/DNApartial heteroduplex involves the following steps: (i) formation of acomposite primer extension product; and (ii) formation of a secondprimer extension product by primer extension along the first primerextension product. For example, in some embodiments, complex comprisingan RNA/DNA partial heteroduplex is generated as follows: followingrandom hybridization of the composite primer at multiple sites ontemplate polynucleotide strands, a DNA polymerase extends the compositeprimer along the template strand generating a composite primer extensionproduct that is complementary to the polynucleotide template strand.Primer extension extends to and displaces strands being extended fromprimers hybridized at upstream sites on the template. Thus, as notedabove, an aspect of the invention is displacement of intervening primerextension product during primer extension of composite primershybridized at downstream site(s) on the template, whereby compositeprimer extension products are displaced from template polynucleotide.FIG. 3 illustrates primer extension from composite primers that arehybridized at multiple sites on a template strand, where a compositeprimer extension products is being displaced by primer extension from acomposite primer hybridized at a downstream site(s) on template strand.

Displaced composite primer product comprises the composite primersequence at the 5′ end, including the 5′ RNA portion. Although forconvenience, reference is made only to “a” composite primer extensionproduct, it is understood that a multiplicity of composite primerextension products are generated the complement of a multiplicity oftemplate polynucleotide sequences appended (via extension) to thesequence of the composite primer. FIG. 4 shows a collection of compositeprimer extension products comprising composite primer 1 linked (byextension) to sequences comprising the complement of a multiplicity oftarget polynucleotide sequences.

Using displaced composite primer extension product as a template, asecond primer extension product complementary to the first primerextension product is generated by extension by a DNA-dependent DNApolymerase along the DNA portion of the composite primer extensionproduct, and extension by a RNA-dependent DNA polymerase along the 5′RNA portion of the composite primer extension product, generating adouble stranded complex comprising a RNA/DNA complex at the end.Generation of second primer extension product may be random primed usingthe composite primer, as depicted in FIG. 5. Alternatively, secondprimer extension product may be primed by the 3′ end of a differentcomposite primer extension product. Additional embodiments in whichsecond strand production is primed by exogenous (added) primers and/orby fragments of template RNA (endogenous primers) are described below.

(b) Single Primer Isothermal Amplification Using a Complex Comprising anRNA/DNA Partial Heteroduplex as a Template

In a second phase of the methods, termed single primer isothermalamplification, the complex comprising an RNA/DNA partial heteroduplex isa substrate for further amplification as follows: an enzyme whichcleaves RNA sequence from an RNA/DNA hybrid (such as RNase H) cleavesRNA from the partial heteroduplex, leaving a partially double strandedpolynucleotide complex comprising a 3′ single stranded DNA sequence. The3′ single stranded sequence (formed by cleavage of RNA in the complexcomprising an RNA/DNA partial heteroduplex) is generally the complementof the RNA in the composite primer, and thus forms a specific bindingsite for a composite primer (which may or may not be the same as thefirst composite primer). Extension of a bound composite primer by aDNA-dependent DNA polymerase produces a primer extension product, whichdisplaces the previously bound cleaved primer extension product, wherebypolynucleotide (generally, DNA) product accumulates. See, for example,U.S. Pat. Nos. 6,251,639 and 6,692,918. FIG. 6 shows amplification ofDNA product using a composite primer and a complex comprising a RNA/DNApartial heteroduplex as a template for further amplification.

Amplification using a complex comprising an RNA/DNA partial heteroduplexas a template for further amplification (also termed single primerisothermal amplification) generally occurs under conditions permittingcomposite primer hybridization, primer extension, cleavage of RNA froman RNA/DNA hybrid and strand displacement. In so far as the compositeprimer hybridizes to the 3′ single stranded portion (of the partiallydouble stranded polynucleotide which is formed by cleaving RNA in thecomplex comprising an RNA/DNA partial heteroduplex) comprising,generally, the complement of at least a portion of the composite primersequence, composite primer hybridization may be under conditionspermitting specific hybridization. Thus, in some embodiments, thereactions conditions permit stringent hybridization (i.e., hybridizationof sequences that are generally complementary). As is evident from thedescription herein, in other embodiments, the reaction conditions areless stringent (i.e., permit hybridization of sequences that are lessthan fully complementary).

Generally, the methods of the invention result in amplification of amultiplicity, a large multiplicity, or a very large multiplicity oftemplate polynucleotide sequences. In some embodiments, essentially allof the template polynucleotide present in the initial sample (e.g., allof the mRNA or all of the genomic DNA) is amplified. In otherembodiments, at least 50, at least 100, at least 200, at least 300, ormore distinct sequences (such as a gene or other subsegment of apolynucleotide, a marker (such as a SNP or other polymorphism) areamplified, as assessed, e.g., by analysis of marker sequences known tobe present in the template sample under analysis, using methods known inthe art. Template polynucleotide sequences that are amplified may bepresent on the same polynucleotide (e.g., a chromosome or portion of achromosome for genomic DNA template or on the same RNA for RNA template)or on different template polynucleotides (e.g., different chromosome orportions of chromosomes for DNA template, or different RNAs for RNAtemplate). Although, amplification of genomic DNA is exemplified herein,it will be understood by those of skill in the art, however, that theglobal amplification methods of the invention are suitable foramplification of any pool or subset of polynucleotides.

For convenience, reference is made to a polynucleotide (generally, DNA)product. It is understood that amplified product generally is a mixtureof sense and antisense copies of a given template polynucleotide. Forexample, if the template polynucleotide is double stranded DNA, theamplification product will correspond to each strand. If the templatepolynucleotide is single stranded (e.g., RNA or single stranded DNA),amplification product will generally be produced that is the copy oftemplate polynucleotide (sense copy) and the complement of the templatepolynucleotide (antisense copy). The amplification product of differentsenses can be annealed to form a double stranded (or partially doublestranded) complex, or can be prevented from annealing (or subsequentlydenatured) to produce a mixture of single stranded amplificationproducts. The amplified products may be of differing lengths.

As is evident from the description and shown in the example, the methodsof the invention are composite-primer dependent. That is, amplificationis not observed in the absence of the composite primer.

As illustrated in these embodiments, all steps are isothermal (in thesense that thermal cycling is not required), although the temperaturesfor each of the steps may or may not be the same. It is understood thatvarious other embodiments may be practiced, given the generaldescription provided above. For example, as described and exemplifiedherein, certain steps may be performed as temperature is changed (e.g.,raised, or lowered).

For simplicity, the methods of the invention are described as twodistinct steps or phases, above. It is understood that the two phasesmay occur simultaneously in some embodiments (for example, if the enzymethat cleaves RNA from RNA/DNA hybrid is included in the first reactionmixture). In other embodiments, step (b) may be initiated by addition ofan enzyme that cleaves RNA from an RNA/DNA hybrid (e.g., ribonuclease,such as RNase H), and optionally, a DNA-dependent DNA polymerase, asshown in Example 1. In this embodiment, addition of an enzyme thatcleaves RNA from an RNA/DNA hybrid permits further amplification usingthe complex comprising an RNA/DNA partial heteroduplex as a template(i.e., step (b), above). It is understood, however, that primerextension (and strand displacement) along template polynucleotide strandfrom random primed composite primer(s) may continue during single primerisothermal amplification.

Although generally only one composite primer is described above, it isfurther understood that the amplification methods may be performed inthe presence of two or more different composite primers that randomlyprime template polynucleotide. In addition, the amplificationpolynucleotide products of two or more separate amplification reactionsconducted using two or more different composite primers that randomlyprime template polynucleotide can be combined. In addition, it isunderstood that different composite primers can be used in step (a)(i.e., random priming of template polynucleotide) and step (b) (i.e.,single primer isothermal amplification). In this instance, the differentcomposite primer comprises sequences hybridizable to the 3′ singlestranded DNA portion of the partially double stranded complex (which isgenerated by cleaving RNA from the complex comprising a RNA/DNA partialheteroduplex). Generally, the second composite primer comprisessequences overlapping with the first composite primer.

Amplification Using a Composite Primer that Hybridizes to a Multiplicityof Template Polynucleotide Sites and Auxiliary Primers

In another aspect of the invention, auxiliary primers are present in thereaction mixture comprising template polynucleotide, composite primer,DNA-dependent DNA polymerase and RNA-dependent DNA polymerase. As usedherein, “auxiliary primers” refers to a population of random orpartially randomized primers. An example of auxiliary primers is therandom hexamer primers used in Example 1. Inclusion of auxiliary primers(i.e., population of different random primers) during the amplificationis believed to enhance the efficiency of production of and/or targetcoverage of the amplification product.

In some embodiments, the methods of the invention work as follows: (a)incubating a reaction mixture, said reaction mixture comprising acomposite primer as described herein; auxiliary primers; a templatepolynucleotide, DNA-dependent DNA polymerase, and RNA-dependent DNApolymerase (which may be present as a single enzyme comprising bothactivities), wherein the incubation is under conditions suitable forrandom composite primer hybridization, auxiliary primer hybridization,primer extension, and strand displacement, whereby a complex comprisingan RNA/DNA partial heteroduplex is generated; and (b) incubating areaction mixture, said reaction mixture comprising the reaction productsfrom step (a) (or an aliquot thereof); a composite primer (which may bethe same as the composite primer of step (a) or may be a differentcomposite primer); a DNA-dependent DNA polymerase; optionally, auxiliaryprimers; and an enzyme that cleaves RNA from a RNA/DNA hybrid; whereinthe incubation is under conditions that permit primer hybridization,primer extension, RNA cleavage from an RNA/DNA heteroduplex, anddisplacement of the primer extension product from the complex when itsRNA is cleaved and another composite primer binds to the template and isextended, whereby multiple copies of a polynucleotide template sequenceare generated.

Inclusion of auxiliary primers (i.e., a population of different randomprimers) during the amplification is believed to enhance the efficiencyof production of and/or coverage of template polynucleotide. Withoutbeing bound by theory, it is believed that primer extension of auxiliaryprimers increases displacement of composite primer extension productfrom template polynucleotide and/or primes generation of second primerextension product. FIG. 7 illustrates primer extension from compositeprimers and auxiliary primers that are hybridized at multiple sites on atemplate strand. FIG. 8 illustrates generation of a second primerextension product primed by auxiliary primers hybridized to compositeprimer extension product.

Although for simplicity, use of auxiliary primers is described only inthe first phase, random composite primer hybridization (i.e., step (a)),it is evident that auxiliary primers may be present in the reactionmixture for the second phase of the methods, single primer isothermalamplification (i.e., step (b)).

As is evident from the description and shown in the example, the methodsof the invention are composite-primer dependent. That is, amplificationis not observed in the absence of the composite primer.

Amplification Using a Composite Primer that Hybridizes to a Multiplicityof Template RNA Sites and Auxiliary Primers

In another aspect of the invention, auxiliary primers are present in thereaction mixture comprising template RNA, composite primer,DNA-dependent DNA polymerase and RNA-dependent DNA polymerase. As usedherein, “auxiliary primers” refers to a population of random orpartially randomized primers. Inclusion of auxiliary primers (i.e.,population of different random primers) during the amplification isbelieved to enhance the efficiency of production of and/or targetcoverage of the amplification product.

In some embodiments, the methods of the invention operate as follows:(a) incubating a reaction mixture, said reaction mixture comprising acomposite primer as described herein; auxiliary primers; a template RNA,DNA-dependent DNA polymerase, and RNA-dependent DNA polymerase (whichmay be present as a single enzyme comprising both activities), whereinthe incubation is under conditions suitable for random composite primerhybridization, auxiliary primer hybridization, primer extension, andstrand displacement, whereby a complex comprising a RNA/DNA partialheteroduplex is generated; and (b) incubating a reaction mixture, saidreaction mixture comprising the reaction products from step (a) (or analiquot thereof); a composite primer (which may be the same as thecomposite primer of step (a) or may be a different composite primer); aDNA-dependent DNA polymerase; optionally, auxiliary primers; and anenzyme that cleaves RNA from an RNA/DNA hybrid; wherein the incubationis under conditions that permit primer hybridization, primer extension,RNA cleavage from an RNA/DNA heteroduplex, and displacement of theprimer extension product from the complex when its RNA is cleaved andanother composite primer binds to the template and is extended, wherebymultiple copies of a polynucleotide template sequence are generated.

Inclusion of auxiliary primers (i.e., a population of different randomprimers) during the amplification is believed to enhance the efficiencyof production of and/or coverage of template RNA. Without being bound bytheory, it is believed that primer extension of auxiliary primersincreases displacement of composite primer extension product fromtemplate RNA and/or primes generation of second primer extensionproduct. FIG. 7 illustrates primer extension from composite primers andauxiliary primers that are hybridized at multiple sites on a templatestrand. FIG. 8 illustrates generation of a second primer extensionproduct primed by auxiliary primers hybridized to composite primerextension product.

Although for simplicity, use of auxiliary primers is described only inthe first phase, random composite primer hybridization (i.e., step (a)),it is evident that auxiliary primers may be present in the reactionmixture for the second phase of the methods, single primer isothermalamplification (i.e., step (b)).

As is evident from the description and shown in the example, the methodsof the invention are composite-primer dependent. That is, amplificationis not observed in the absence of the composite primer.

Components and Reaction Conditions Used in the Methods of the Invention

Template Nucleic Acid

The nucleic acid (NA) target to be amplified includes nucleic acids fromany source in purified or unpurified form, which can be DNA (dsDNA andssDNA) or RNA, including tRNA, mRNA, rRNA, mitochondrial DNA and RNA,chloroplast DNA and RNA, DNA-RNA hybrids, or mixtures thereof, genes,chromosomes, plasmids, the genomes of biological material such asmicroorganisms, e.g., bacteria, yeasts, viruses, viroids, molds, fungi,plants, animals, humans, and fragments thereof. Preferred targetpolynucleotide includes DNA (e.g., genomic DNA, including human genomicDNA, and mammalian genomic DNA (such as mouse, rat,))and RNA (e.g.,mRNA, ribosomal RNA, and total RNA). It should be understood thattemplate RNA includes coding and non-coding RNA. The sequences can benaturally occurring or recombinant nucleic acid targets, includingcloned nucleic fragments of interest.

The target nucleic acid can be only a minor fraction of a complexmixture such as a biological sample and can be obtained from variousbiological material by procedures well known in the art. Nucleic acidcan be obtained from sources containing very small quantities of nucleicacid, such a single cells, small numbers of cells, patient samples,forensic samples, and archeological samples. Obtaining and purifyingnucleic acids use standard techniques in the art, including methodsdesigned to isolate one or a very small number of cells, such a cellsorting or laser capture micro-dissection. The methods of the inventionare particularly suited for use with genomic DNA (e.g., human and othermammalian genomic DNA), as well as RNA (e.g., total RNA or mRNAsamples). Amplification of an RNA target may be accomplished by initialcDNA synthesis, as known in the art, followed by amplification from thecDNA template.

The target polynucleotide(s) can be known or unknown and may containmore than one desired specific nucleic acid sequence of interest, eachof which may be the same or different from each other. If the targetpolynucleotide is double stranded (e.g., double stranded DNA or a doublestranded DNA/RNA hybrid, such as is produced by first strand cDNAsynthesis), the target may first be treated to render it single stranded(e.g., by denaturation or by cleavage of the RNA portion of a DNA/RNAhybrid). Denaturation may also be carried out to remove secondarystructure present in a single stranded target molecule (e.g., RNA). Insome cases, double stranded DNA target polynucleotide may be firstcleaved by one or more restriction endonuclease enzymes.

When the target polynucleotide is DNA, the initial step of theamplification of a target nucleic acid sequence is rendering the targetsingle stranded. If the target nucleic acid is a double stranded (ds)DNA, the initial step can be target denaturation. The denaturation stepmay be thermal denaturation or any other method known in the art, suchas alkali treatment. If the target nucleic acid is present in an DNA-RNAhybrid, the initial step can be denaturation of the hybrid to obtain aDNA, or removal of the RNA strand using other means known in the art,such as thermal treatment, digestion with an enzyme that cleaves RNAfrom an RNA/DNA hybrid (such as RNase H) or alkali treatment, togenerate single stranded DNA. When the target is RNA, the initial stepmay be the synthesis of a single stranded cDNA. Techniques for thesynthesis of cDNA from RNA are known in the art, and include reversetranscription of RNA strand using a primer that binds to a specifictarget, such as the poly-A tail of eukaryotic mRNAs or other specific orconsensus sequences. In addition, reverse transcription can be primed bya population of degenerate or partially degenerate primers. First strandcDNA can be separated from the complex of RNA and first strand cDNA asdescribed herein.

RNAs can be from any source in purified or unpurified form, which can beRNA such as total RNA, tRNA, mRNA, rRNA, mitochondrial RNA, chloroplastRNA, DNA-RNA hybrids, or mixtures thereof, from any source and/orspecies, including human, animals, plants, and microorganisms such asbacteria, yeasts, viruses, viroids, molds, fungi, plants, and fragmentsthereof. It is understood that the RNA can be coding or noncoding RNA(such as untranslated small RNAs). RNAs can be obtained and purifiedusing standard techniques in the art. Use of a DNA target (includinggenomic DNA target) would require initial transcription of the DNAtarget into RNA form, which can be achieved using methods disclosed inKurn, U.S. Pat. No. 6,251,639 B1, and by other techniques (such asexpression systems) known in the art. Thus, RNA template can be itselfgenerated from a DNA source (such as genomic DNA), using methods knownin the art, including Kurn, U.S. Pat. No. 6,251,639. RNA copies ofgenomic DNA would generally include untranscribed sequences generallynot found in mRNA, such as introns, regulatory and control elements,etc. RNA targets may also be generated from cloned genomic DNA sequencesthat can be subjected to in vitro transcription. Use of a DNA-RNA hybridwould require denaturation of the hybrid to obtain a single strandedRNA, denaturation followed by transcription of the DNA strand to obtainan RNA, or other methods known in the art such as digestion with anRNAse H to generate single stranded DNA.

Composite Primer

The methods of the invention employ a composite primer that is composedof RNA and DNA portions. We have observed that suitable compositeprimers show partial nucleic acid sequence homology to a multiplicity ofgenomic DNA sequences, particularly in the 3′ sequences of the compositeprimer, when analyzed using standard nucleic acid comparison algorithms.For example, composite primer sequence can be used as a query sequencein Blast, to search the human genomic DNA database (or other suitabledatabase, such as a mammalian genomic DNA database). Generally, thesearch is performed using search parameters suitable for identificationof partial or “low stringency” alignments, generally the least stringentconditions provided by the program. Such parameters are known in the artand include use of the NCBI Blast program for searching “short, nearlyexact matches”, with word size=7 (conditions permitting as few as 7consecutive nucleotide perfect matches at any position in the primersequence). See, e.g.,http://www.ncbi.nlm.nih.gov/blast/Blast.cgi?ALIGNMENTS=50&ALIGNMENT_VIEW=Pairwise&AUTO_FORMAT=Semiauto&CLIENT=web&DATABASE=nr&DESCRIPTIONS=100&ENTREZ_QUERY=>(none)&EXPECT=1000&FORMAT_BLOCK_ON_RESPAGE=None&FORMAT_ENTREZ_QUERY=(none)&FORMAT_OBJECT=Alignment&FORMAT_TYPE=HTML&LAYOUT=TwoWindows&NCBI_GI=on&PAGE=Nucleotides&PROGRAM=blastn&SERVICE=plain&SET_DEFAULTS.x=16&SET_DEFAULTS.y=8&SHOW_OVERVIEW=on&WORD_SIZE=7&END_OF_HTTPGET=Yes.Composite primers useful in the methods of the invention (i.e., thatrandomly hybridize to template polynucleotide) generally exhibit highpartial homology rate with genomic DNA sequences, for example homologyof stretches of 7 nucleotides with about 100 genomic DNA sequences, withabout 70% of the hits located at the 3′ end of the composite primer. Acomposite primer with a very unique sequence (i.e., low levels ofhomology with target genomic DNA sequences) did not function efficientlyin the methods of the invention when used with genomic DNA template.

As is evident from the discussion above, reference to a primer thatbinds (hybridizes to) a sequence (or template) encompasses embodimentsin which at least a portion of the primer is hybridized, embodiments inwhich two (or more portions) of the primer are hybridized (separated byunhybridized (looped out) portions of the primer), and embodiments inwhich the entire primer is hybridized. In certain embodiments, a5′-portion, commonly the 5′-most portion, of the composite primer isdesigned such that the particular 5′-portion it is not expected torandomly hybridize to template polynucleotide (composite primers of thisconfiguration are referred to as “tailed” primers, in reference to the‘tail’ of unhybridized primer). In some embodiments, the tail portion ofthe composite primer is the entire 5′ RNA portion of the compositeprimer. Thus, according to the methods of the invention, only a portionof the 3′-end of the composite primer must be hybridized in order forinitiation of primer extension by DNA polymerase. In some embodiments,for example, only 2, 3, 4, 5, 6, 7 or more nucleotides of the 3′ end ofthe primer must hybridize in order for primer extension to be initiated.It is understood that hybridization of the 3′-most portion of thecomposite primer may be stabilized to various extents by furtherhybridization of another portion of the primer (with or without loopingout of intervening primer portions). A DNA polymerase can be includedduring primer hybridization to enhance (e.g., stabilize) hybridizationof composite primer by initiation of primer extension, and thus,stabilization of primer hybridization.

We have also observed that composite primers that are suitable for usein the present methods can be identified by conducting single primerisothermal amplification as described in Kurn, U.S. Pat. No. 6,251,639,using the composite primer under high stringency conditions using agenomic DNA template, and observing the presence of a smear of reactionproducts as visualized, for example, on a gel. Preferably, the genomicDNA does not contain a sequence(s) that is complementary to thecomposite primer. Production of a “smear” of reaction products, i.e.,generation of a complex mixture of product of multiple molecularweights, visible on a gel as a smear, indicates that the compositeprimer is randomly priming genomic DNA amplification.

In another example, single primer isothermal amplification of a specificsynthetic target oligonucleotide (e.g., a target oligonucleotidecomprising a specific target for composite primer hybridization) isconducted at high stringency in the presence or absence of genomic DNAtemplate (e.g., 1-100 ng of human genomic DNA). Composite primers thatare suitable for the methods of the invention will demonstrate a strongeffect of genomic DNA on the efficiency of the amplification of thespecific synthetic target, resulting in about a 100-fold or greaterreduction of amplification efficiency as compared with amplificationefficiency conducted in the absence of genomic DNA.

Random composite primer hybridization and/or generation of compositeprimer extension product is promoted by use of conditions designed topermit random (non-specific) primer hybridization. Such conditions arewell known in the art, and are further discussed below, and include:decreased stringency during primer hybridization and/or first strandsynthesis (including reduced temperature and/or buffer conditions ofreduced stringency, such as reduced ionic strength); composite primerselection and/or design (discussed further herein); composite primer andtemplate concentration, presence or absence of an agent that stabilizesa 3′ hybridized primer (such as a DNA polymerase), and presence orabsence of agents such as DMSO that lower the temperature requirementsfor stable hybridization. It is understood that the selection ofreaction conditions may be used to control the frequency of compositeprimer hybridization, and thus control coverage and/or representation oftemplate polynucleotide sequences in amplification product.

Generally, the composite primer is also designed so that there is noprimer-dimer formation capability, as determined using softwareroutinely available to one of ordinary skill in the art, e.g. OligoPrimer Analysis Software from Molecular Biology Insight, and referencestherein. One of skill in the art will understand that other factorsaffect nucleic acid hybridization affinities. For example, any and allof the guanosine-cytosine content of the primer-target and primer-primerduplexes, minor groove binders, O-methylation or other modification ofnucleotides, temperature, and salt are potentially important factors inconstructing primers with the requisite differences in binding energies.Another factor in designing and constructing primers is the free energyparameters of hybridization of given sequences under a given set ofhybridization conditions. The free energy parameters for the formationof a given hybrid may be calculated by methods known in the art (see,e.g., Tinoco et al. Nature (1973) 246:40-41 and Freier et al., Proc.Natl. Acad. Sci. USA (1986) 83:9373-9377; computer programs, e.g., OligoPrimer Analysis Software from Molecular Biology Insight, and referencestherein), and it is possible to predict, for a given oligonucleotidetemplate, primer sequences for which the required free energy changesfor formation of various complexes will be met.

The primers should be extendable by DNA polymerase. Generation ofprimers suitable for extension by polymerization is well known in theart, such as described in PCT Pub. No. WO99/42618 (and references citedtherein). Generally, the primer should permit high efficiency ofamplification of a synthetic target that contains a specific primertarget binding site (e.g., the complementary sequence to the primer),for example, permitting amplification of about 10⁶ to 10⁹ using methodsdescribed in Kurn, U.S. Pat. No. 6,251,639. The composite primer isdesigned such that subsequent displacement of the primer extensionproduct by binding of a new (additional) composite primer and theextension of the new primer by the polymerase can be achieved. Inaddition, cleavage of the RNA portion of the primer extension productleads to generation of amplification product which is not a substratefor amplification by the composite primer. It is understood that, in thefollowing section that generally describes aspects of the compositeprimers used in the methods of the invention, characteristics describedmay be applicable to the primers if used for hybridizing and initiatingthe polynucleotide amplification (production of composite extensionproduct) and/or for single primer isothermal amplification as describedherein.

In some embodiments, a first composite primer is used in the methods ofthe invention, including those steps which involve single primerisothermal amplification (i.e., phase (b)). In other embodiments, afirst and second, different, composite primer are used in the methods ofthe invention. The second composite primer is used for the single primerisothermal amplification step, and may comprise some or all of thesequence of the first composite primer, and the first composite primermay comprise some or all of the sequence of the second composite primer.In some embodiments, the second composite primer comprises a differentsequence than the first composite primer.

For use in single primer isothermal amplification and/or compositeprimer extension product formation, a composite primer comprises atleast one RNA portion that is capable of (a) binding (hybridizing) to asequence on the single stranded portion of the complex (formed bycleavage of RNA in the complex comprising a RNA/DNA partialheteroduplex) (in some embodiments, on second primer extension product)independent of hybridization of the DNA portion(s) to a sequence on thesame single stranded portion; and (b) being cleaved with an agent suchas a ribonuclease when hybridized to the single stranded portion. Thecomposite primers bind to the single stranded portion, and are extendedby DNA polymerase to form a RNA/DNA partial heteroduplex in which onlythe RNA portion of the primer is cleaved upon contact with an agentwhich cleaves RNA in an RNA/DNA hybrid, such as an enzyme, such as aribonuclease (such as RNase H), while the composite primer extensionproduct remains intact, thus enabling annealing of another compositeprimer.

When used for the single primer isothermal amplification describedherein, the composite primers also comprise a 3′ DNA portion that iscapable of hybridization to a sequence on the 3′ single stranded portionof the complex such that its hybridization to the 3′ single strandedportion is favored over that of the nucleic acid strand that isdisplaced from the complex by the DNA polymerase. Such primers can berationally designed based on well known factors that influence nucleicacid binding affinity, such as sequence length and/or identity, as wellas hybridization conditions. In a preferred embodiment, hybridization ofthe 3′ DNA portion of the composite primer to its complementary sequencein the complex (e.g., in the second primer extension product) favoredover the hybridization of the homologous sequence in the 5′ end of thedisplaced strand to the composite primer extension product.

The composite primer comprises a combination of RNA and DNA (seedefinition above), with the 3′-end nucleotide being a nucleotidesuitable for nucleic acid extension. The 3′-end nucleotide can be anynucleotide or analog that when present in a primer, is extendable by aDNA polymerase. Generally, the 3′-end nucleotide has a 3′-OH. Suitableprimers include those that comprise at least one portion of RNA and atleast one portion of DNA. For example, composite primers can comprise a5′-RNA portion and a 3′-DNA portion (in which the RNA portion isadjacent to the 3′-DNA portion); or 5′- and 3′-DNA portions with anintervening RNA portion. Accordingly, in one embodiment, the compositeprimer comprises a 5′ RNA portion and a 3′-DNA portion, preferablywherein the RNA portion is adjacent to the 3′-DNA portion. In anotherembodiment, the composite primer comprises 5′- and 3′-DNA portions withat least one intervening RNA portion (i.e., an RNA portion between thetwo DNA portions). In yet another embodiment, the composite primer ofthe invention comprises a 3′-DNA portion and at least one interveningRNA portion (i.e., an RNA portion between DNA portions).

The length of an RNA portion in a composite primer comprising a 3′-DNAportion and an RNA portion can be preferably from about 1 to about 50,more preferably from about 3 to about 20, even more preferably fromabout 4 to about 15, and most preferably from about 5 to about 10nucleotides. In some embodiments of a composite primer comprising a3′-DNA portion and an RNA portion, an RNA portion can be at least aboutany of 1, 3, 4, 5 nucleotides, with an upper limit of about any of 10,14, 15, 20, 25, 3, 50 nucleotides. In certain embodiments, the compositeprimer has an RNA portion of about 14 or about 20 nucleotides.

The length of the 5′-RNA portion in a composite primer comprising a5′-RNA portion and a 3′-DNA portion can be preferably from about 3 toabout 50 nucleotides, more preferably from about 5 to about 20nucleotides, even more preferably from about 7 to about 18 nucleotides,preferably from about 8 to about 17 nucleotides, and most preferablyfrom about 10 to about 15 nucleotides. In other embodiments of acomposite primer comprising a 5′-RNA portion and a 3′-DNA portion, the5′-RNA portion can be at least about any of 3, 5, 7, 8, 10 nucleotides,with an upper limit of about any of 14, 15, 17, 18, 20, 50 nucleotides.In certain embodiments, the composite primer has an RNA portion of about14 or about 20 nucleotides.

In embodiments of a composite primer comprising a 5′-RNA portion and a3′-DNA portion further comprising non-5′-RNA portion(s), a non-5′-RNAportion can be preferably from about 1 to about 7 nucleotides, morepreferably from about 2 to about 6 nucleotides, and most preferably fromabout 3 to about 5 nucleotides. In certain embodiments of a compositeprimer comprising a 5′-RNA portion and a 3′-DNA portion furthercomprising non-5′-RNA portion(s), a non-5′-RNA portion can be at leastabout any of 1, 2, 3, 5, with an upper limit of about any of 5, 6, 7, 10nucleotides.

In embodiments of a composite primer comprising a 5′-RNA portion and a3′-DNA portion, in which the 5′-RNA portion is adjacent to the 3′-DNAportion, the length of the 5′-RNA portion can be preferably from about 3to about 50 nucleotides, more preferably from about 5 to about 20nucleotides, even more preferably from about 7 to about 18 nucleotides,preferably from about 8 to about 17 nucleotides, and most preferablyfrom about 10 to about 15 nucleotides. In certain embodiments of acomposite primer comprising a 5′-RNA portion and a 3′-DNA portion, inwhich the 5′-RNA portion is adjacent to the 3′-DNA portion, the 5′-RNAportion can be at least about any of 3, 5, 7, 8, 10 nucleotides, with anupper limit of about any of 14, 15, 17, 18, 20, 50 nucleotides. Incertain embodiments, the composite primer has an RNA portion of about 14or about 20 nucleotides.

The length of an intervening RNA portion in a composite primercomprising 5′- and 3′-DNA portions with at least one intervening RNAportion can be preferably from about 1 to about 7 nucleotides, morepreferably from about 2 to about 6 nucleotides, and most preferably fromabout 3 to about 5 nucleotides. In some embodiments of a compositeprimer comprising 5′- and 3′-DNA portions with at least one interveningRNA portion, an intervening RNA portion can be at least about any of 1,2, 3, 5 nucleotides, with an upper limit of about any of 5, 6, 7, 10nucleotides. The length of an intervening RNA portion in a compositeprimer comprising a 3′-DNA portion and at least one intervening RNAportion can be preferably from about 1 to about 7 nucleotides, morepreferably from about 2 to about 6 nucleotides, and most preferably fromabout 3 to about 5 nucleotides. In some embodiments of a compositeprimer comprising a 3′-DNA portion and at least one intervening RNAportion, an intervening RNA portion can be at least about any of 1, 2,3, 5 nucleotides, with an upper limit of about any of 5, 6, 7, 10nucleotides. In a composite primer comprising a 3′-DNA portion and atleast one intervening RNA portion, further comprising a 5′-RNA portion,the 5′-RNA portion can be preferably from about 3 to about 25nucleotides, more preferably from about 5 to about 20 nucleotides, evenmore preferably from about 7 to about 18 nucleotides, preferably fromabout 8 to about 17 nucleotides, and most preferably from about 10 toabout 15 nucleotides. In some embodiments of a composite primercomprising a 3′-DNA portion and at least one intervening RNA portion,further comprising a 5′-RNA portion, the 5′-RNA portion can be at leastabout any of 3, 5, 7, 8, 10 nucleotides, with an upper limit of aboutany of 15, 17, 18, 20 nucleotides.

The length of the 3′-DNA portion in a composite primer comprising a3′-DNA portion and an RNA portion can be preferably from about 1 toabout 20, more preferably from about 3 to about 18, even more preferablyfrom about 5 to about 15, and most preferably from about 7 to about 12nucleotides. In some embodiments of a composite primer comprising a3′-DNA portion and an RNA portion, the 3′-DNA portion can be at leastabout any of 1, 3, 5, 7, 10 nucleotides, with an upper limit of aboutany of 10, 12, 15, 18, 20, 22 nucleotides. In one embodiment, thecomposite primer has a 3′-DNA portion of about 7 nucleotides.

The length of the 3′-DNA portion in a composite primer comprising a5′-RNA portion and a 3′-DNA portion can be preferably from about 1 toabout 20 nucleotides, more preferably from about 3 to about 18nucleotides, even more preferably from about 5 to about 15 nucleotides,and most preferably from about 7 to about 12 nucleotides. In someembodiments of a composite primer comprising a 5′-RNA portion and a3′-DNA portion, the 3′ DNA portion can be at least about any of 1, 3, 5,7, 10 nucleotides, with an upper limit of about any of 10, 12, 15, 18,20, 22 nucleotides. In one embodiment, the composite primer has a 3′-DNAportion of about 7 nucleotides.

In embodiments of a composite primer comprising a 5′-RNA portion and a3′-DNA portion, further comprising non-3′-DNA portion(s), a non-3′-DNAportion can be preferably from about 1 to about 10 nucleotides, morepreferably from about 2 to about 8 nucleotides, and most preferably fromabout 3 to about 6 nucleotides. In some embodiments of a compositeprimer comprising a 5′-RNA portion and a 3′-DNA portion, furthercomprising non-3′-DNA portion(s), a non-3′-DNA portion can be at leastabout any of 1, 2, 3, 5 nucleotides, with an upper limit of about any of6, 8, 10, 12 nucleotides.

In embodiments of a composite primer comprising a 5′-RNA portion and a3′-DNA portion in which the 5′-RNA portion is adjacent to the 3′-DNAportion, the length of the 3′-DNA portion can be preferably from about 1to about 20 nucleotides, more preferably from about 3 to about 18nucleotides, even more preferably from about 5 to about 15 nucleotides,and most preferably from about 7 to about 12 nucleotides. In certainembodiments of the primer comprising a 5′-RNA portion and a 3′-DNAportion in which the 5′-RNA portion is adjacent to the 3′-DNA portion,the 3′-DNA portion can be at least about any of 1, 3, 5, 7, 10nucleotides, with an upper limit of about any of 10, 12, 15, 18, 20, 22nucleotides. In one embodiment, the composite primer has a 3′-DNAportion of about 7 nucleotides.

The length of a non-3′-DNA portion in a composite primer comprising 5′-and 3′-DNA portions with at least one intervening RNA portion can bepreferably from about 1 to about 10 nucleotides, more preferably fromabout 2 to about 8 nucleotides, and most preferably from about 3 toabout 6 nucleotides. In some embodiments of a primer comprising 5′- and3′-DNA portions with at least one intervening RNA portion, a non-3′-DNAportion can be at least about any of 1, 2, 3, 5 nucleotides, with anupper limit of about any of 6, 8, 10, 12 nucleotides.

The length of the 3′-DNA portion in a composite primer comprising 5′-and 3′-DNA portions with at least one intervening RNA portion can bepreferably from about 1 to about 20 nucleotides, more preferably fromabout 3 to about 18 nucleotides, even more preferably from about 5 toabout 15 nucleotides, and most preferably from about 7 to about 12nucleotides. In some embodiments of a composite primer comprising 5′-and 3′-DNA portions with at least one intervening RNA portion, the3′-DNA portion can be at least about any of 1, 3, 5, 7, 10 nucleotides,with an upper limit of about any of 10, 12, 15, 18, 20, 22 nucleotides.In one embodiment, the composite primer has a 3′-DNA portion of about 7nucleotides.

The length of a non-3′-DNA portion (i.e., any DNA portion other than the3′-DNA portion) in a composite primer comprising a 3′-DNA portion and atleast one intervening RNA portion can be preferably from about 1 toabout 10 nucleotides, more preferably from about 2 to about 8nucleotides, and most preferably from about 3 to about 6 nucleotides. Insome embodiments of a composite primer comprising a 3′-DNA portion andat least one intervening RNA portion, a non-3′-DNA portion can be atleast about any of 1, 3, 5, 7, 10 nucleotides, with an upper limit ofabout any of 6, 8, 10, 12 nucleotides. The length of the 3′-DNA portionin a composite primer comprising a 3 ′-DNA portion and at least oneintervening RNA portion can be preferably from about 1 to about 20nucleotides, more preferably from about 3 to about 18 nucleotides, evenmore preferably from about 5 to about 15 nucleotides, and mostpreferably from about 7 to about 12 nucleotides. In some embodiments ofa composite primer comprising a 3′-DNA portion and at least oneintervening RNA portion, the 3′-DNA portion can be at least about any of1, 3, 5, 7, 10 nucleotides, with an upper limit of about any of 10, 12,15, 18, 20, 22 nucleotides. In one embodiment, the composite primer hasa 3′-DNA portion of about 7 nucleotides. It is understood that thelengths for the various portions can be greater or less, as appropriateunder the reaction conditions of the methods of this invention.

In some embodiments, the 5′-DNA portion of a composite primer includesthe 5′-most nucleotide of the primer. In some embodiments, the 5′-RNAportion of a composite primer includes the 5′ most nucleotide of theprimer. In other embodiments, the 3′-DNA portion of a composite primerincludes the 3′ most nucleotide of the primer. In other embodiments, the3′-DNA portion is adjacent to the 5′-RNA portion and includes the 3′most nucleotide of the primer (and the 5′-RNA portion includes the 5′most nucleotide of the primer).

The total length of the composite primer can be preferably from about 10to about 50 nucleotides, more preferably from about 15 to about 30nucleotides, and most preferably from about 20 to about 25 nucleotides.In some embodiments, the length can be at least about any of 10, 15, 20,25 nucleotides, with an upper limit of about any of 25, 30, 50, 60nucleotides. In certain embodiments, the composite primer is about 21 orabout 27 nucleotides in length. It is understood that the length can begreater or less, as appropriate under the reaction conditions of themethods of this invention.

As described herein, one or more different composite primers may be usedin an amplification reaction.

Auxiliary Primers

“Auxiliary primer” as used herein, are a population of primerscomprising randomized and/or partially-randomized sequences. Auxiliaryprimers are a polynucleotide as described herein, though generally,auxiliary primers are made of DNA. Such random primers are known in theart. An example of auxiliary primers is the population of randomizedhexamer primers shown in Example 1. In some embodiments, the randomprimers may contain natural or non-natural nucleotide(s) that permitnon-specific hybridization in order to increase the number of sequencesto which the random primers may bind. Similarly, abasic sites can beintroduced randomly within the population of random primers, which canpermit non-specific hybridization by stabilizing mismatches betweenprimer and template. The primers should be extendable by DNA polymerase.Generation of primers suitable for extension by polymerization is wellknown in the art, such as described in PCT Pub. No. WO 99/42618 (andreferences cited therein).

In some embodiments, auxiliary primers can be at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 15,at least 18, at least 20, or more nucleotides in length. In someembodiments, a population of primers of differing lengths is used.

DNA Polymerase, and an Agent Capable of Cleaving an RNA-DNA Hybrid

The amplification methods of the invention employ the following enzymes:an RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, and anagent capable of cleaving an RNA strand of an RNA-DNA hybrid (forexample, a ribonuclease such as RNase H). One or more of theseactivities may be found and used in a single enzyme. For example, RNaseH activity may be supplied by an RNA-dependent DNA polymerase (such asreverse transcriptase) or may be provided in a separate enzyme. Reversetranscriptases useful for this method may or may not have RNase Hactivity. Many reverse transcriptases, such as those from avianmyeloblastosis virus (AMV-RT), and Moloney murine leukemia virus(MMLV-RT) comprise more than one activity (for example, polymeraseactivity and ribonuclease activity) and can function in the formation ofthe double stranded cDNA molecules. However, in some instances, it ispreferable to employ a reverse transcriptase which lacks the RNase Hactivity. Reverse transcriptase devoid of RNase H activity are known inthe art, including those comprising a mutation of the wild type reversetranscriptase where the mutation eliminates the RNase H activity. Inthese cases, the addition of an RNase H from other sources, such as thatisolated from E. coli, can be employed for the formation of the doublestranded cDNA. The RNA-dependent DNA polymerase activity andDNA-dependent DNA polymerase activity may be provided by the same enzyme(for example, Bst polymerase), or these activities may be provided inseparate enzymes.

One aspect of the invention is the formation of a complex comprising anRNA/DNA partial heteroduplex. This process generally utilizes theenzymatic activities of an RNA-dependent DNA polymerase, a DNA-dependentDNA polymerase. Generally, RNA in the RNA/DNA partial heteroduplex iscleaved by an agent (such as an enzyme, such as a ribonuclease) capableof cleaving RNA from an RNA/DNA hybrid, generating a 3′ single strandedportion with sequences that are complementary to RNA in a compositeprimer (and thus forming a binding site for a composite primer).

RNA-dependent DNA polymerases for use in the methods and compositions ofthe invention are capable of effecting extension of a primer accordingto the methods of the invention. Accordingly, a preferred RNA-dependentDNA polymerase is one that is capable of extending a nucleic acid primeralong a nucleic acid template that is comprised at least predominantlyof ribonucleotides. Suitable RNA-dependent DNA polymerases for use inthe methods and compositions of the invention include reversetranscriptase and, for example, a DNA polymerase that possesses bothDNA-dependent and RNA-dependent DNA polymerase activity, such as Bst DNApolymerase.

DNA-dependent DNA polymerases for use in the methods and compositions ofthe invention are capable of effecting extension of the composite primeraccording to the methods of the invention. Accordingly, a preferredpolymerase is one that is capable of extending a nucleic acid primeralong a nucleic acid template that is comprised at least predominantlyof deoxynucleotides. The formation of the complex comprising the RNA/DNApartial heteroduplex can be carried out by a DNA polymerase whichcomprises both RNA-dependent DNA polymerase and DNA-dependent DNApolymerase activities (such as Bst DNA polymerase, or a reversetranscriptase). Amplification of an RNA sequence according to methods ofthe invention involves the use of a DNA polymerase that is able todisplace a nucleic acid strand from the polynucleotide to which thedisplaced strand is bound, and, generally, the more strand displacementcapability the polymerase exhibits (i.e., compared to other polymeraseswhich do not have as much strand displacement capability) is preferable.Preferably, the DNA polymerase has high affinity for binding at the3′-end of an oligonucleotide hybridized to a nucleic acid strand.Preferably, the DNA polymerase does not possess substantial nickingactivity. Generally, the DNA polymerase preferably has little or no5′->3′ exonuclease activity so as to minimize degradation of primer, orprimer extension polynucleotides. Generally, this exonuclease activityis dependent on factors such as pH, salt concentration, whether thetemplate is double stranded or single stranded, and so forth, all ofwhich are familiar to one skilled in the art. Mutant DNA polymerases inwhich the 5′->3′ exonuclease activity has been deleted, are known in theart and are suitable for the amplification methods described herein.Mutant DNA polymerases which lack both 5′ to 3′ nuclease and 3′ to 5′nuclease activities have also been described, for example,exo^(−/−)Klenow DNA polymerase. It is preferred that the DNA polymerasedisplaces primer extension products from the template nucleic acid in atleast about 25%, more preferably at least about 50%, even morepreferably at least about 75%, and most preferably at least about 90%,of the incidence of contact between the polymerase and the 5′ end of theprimer extension product. In some embodiments, the use of thermostableDNA polymerases with strand displacement activity is preferred. Suchpolymerases are known in the art, such as described in U.S. Pat. No.5,744,312 (and references cited therein). Preferably, the DNA polymerasehas little to no proofreading activity

Suitable DNA polymerases for use in the methods and compositions of theinvention include those disclosed in U.S. Pat. Nos. 5,648,211 and5,744,312, which include exo⁻ Vent (New England Biolabs), exo⁻ Deep Vent(New England Biolabs), Bst (BioRad), exo⁻ Pfu (Stratagene), Bca(Panvera), sequencing grade Taq (Promega), exo^(−/−)Klenow DNApolymerase, and thermostable DNA polymerases from thermoanaerobacterthermohydrosulfuricus.

The ribonuclease for use in the methods and compositions of theinvention is capable of cleaving ribonucleotides in an RNA/DNA hybrid.Preferably, the ribonuclease cleaves ribonucleotides in an RNA/DNAhybrid regardless of the identity and type of nucleotides adjacent tothe ribonucleotide to be cleaved. It is preferred that the ribonucleasecleaves independent of sequence identity. Examples of suitableribonucleases for the methods and compositions of the invention are wellknown in the art, including ribonuclease H (RNase H), e.g., Hybridase.

As is well known in the art, DNA-dependent DNA polymerase activity,RNA-dependent DNA polymerase activity, and the ability to cleave RNAfrom a RNA/DNA hybrid may be present in different enzymes, or two ormore activities may be present in the same enzyme. Accordingly, in someembodiments, the same enzyme comprises RNA-dependent DNA polymeraseactivity and cleaves RNA from an RNA/DNA hybrid. In some embodiments,the same enzyme comprises DNA-dependent DNA polymerase activity andcleaves RNA from an RNA/DNA hybrid. In some embodiments, the same enzymecomprises DNA-dependent DNA polymerase activity, RNA-dependent DNApolymerase activity and cleaves RNA from an RNA/DNA hybrid. In someembodiments, different enzymes comprise RNA-dependent DNA polymeraseactivity and DNA-dependent DNA polymerase activity. In some embodiments,different enzymes comprise RNA -dependent DNA polymerase activity andcleave RNA from an RNA/DNA hybrid. In some embodiments, differentenzymes comprise DNA-dependent DNA polymerase activity and cleave RNAfrom an RNA/DNA hybrid.

In general, the enzymes used in the methods and compositions of theinvention should not produce substantial degradation of the nucleic acidcomponents of said methods and compositions.

Reaction Conditions and Detection

Appropriate reaction media and conditions for carrying out the methodsof the invention are those that permit nucleic acid amplificationaccording to the methods of the invention. Such media and conditions areknown to persons of skill in the art, and are described in variouspublications, such as U.S. Pat. Nos. 5,554,516; 5,716,785; 5,130,238;5,194,370; 6,090,591; 5,409,818; 5,554,517; 5,169,766; 5,480,784;5,399,491; 5,679,512; and PCT Pub. No. WO99/42618. For example, a buffermay be Tris buffer, although other buffers can also be used as long asthe buffer components are non-inhibitory to enzyme components of themethods of the invention. The pH is preferably from about 5 to about 11,more preferably from about 6 to about 10, even more preferably fromabout 7 to about 9, and most preferably from about 7.5 to about 8.5. Thereaction medium can also include bivalent metal ions such as Mg²⁺ orMn²⁺, at a final concentration of free ions that is within the range offrom about 0.01 to about 15 mM, and most preferably from about 1 to 10mM. The reaction medium can also include other salts, such as KCl orNaCl, that contribute to the total ionic strength of the medium. Forexample, the range of a salt such as KCl is preferably from about 0 toabout 125 mM, more preferably from about 0 to about 100 mM, and mostpreferably from about 0 to about 75 mM. The reaction medium can furtherinclude additives that could affect performance of the amplificationreactions, but that are not integral to the activity of the enzymecomponents of the methods. Such additives include proteins such as BSAor acetylated BSA, single strand binding proteins (for e.g., T4 gene 32protein), and non-ionic detergents such as NP40 or Triton. Reagents,such as DTT, that are capable of maintaining enzyme activities can alsobe included. Such reagents are known in the art. Where appropriate, anRNase inhibitor (such as Rnasin) that does not inhibit the activity ofthe RNase employed in the method can also be included. Any aspect of themethods of the invention can occur at the same or varying temperatures.Preferably, the amplification reactions (particularly, primer extensionother than the composite and second primer extension product synthesissteps, and strand displacement) are performed isothermally, which avoidsthe cumbersome thermocycling process. The amplification reaction iscarried out at a temperature that permits hybridization of theoligonucleotides (primer) of the invention to the templatepolynucleotide and primer extension products, and that does notsubstantially inhibit the activity-of the enzymes employed. Thetemperature can be in the range of 0° C. to about 85° C., about 25° C.to about 85° C., about 30° C. to about 80° C., and about 37° C. to about75° C.

Random priming and/or primer extension and/or isothermal amplificationcan be conducted under conditions of reduced stringency (i.e.,permitting hybridization of sequences that are not fully complementary).For a given set of reaction conditions, the ability of two nucleotidesequences to hybridize with each other is based on the degree ofcomplementarity of the two nucleotide sequences, which in turn is basedon the fraction of matched complementary nucleotide pairs. The morenucleotides in a given sequence that are complementary to anothersequence, the more stringent the conditions can be for hybridization andthe more specific will be the binding of the two sequences. Conversely,the lower the stringency of the conditions for hybridization, the lowerthe complementarity necessary for binding between the hybridizing and/orpartially hybridizing composite primer and template polynucleotide.Decreased stringency is achieved by any one or more of the following:reducing the temperature, decreasing the ratio of cosolvents, loweringthe salt concentration, and the like. Conditions that increase or reducethe stringency of a hybridization reaction are widely known andpublished in the art. See, for example, Sambrook et al. (1989), and inAusubel (1987), supra. Useful hybridization conditions are also providedin, e.g., Tijessen, 1993, Hybridization With Nucleic Acid Probes,Elsevier Science Publishers B.V. and Kricka, 1992, Nonisotopic DNA ProbeTechniques, Academic Press San Diego, Calif. The hybridizationconditions chosen depend on a variety of factors known in the art, forexample the length and type (e.g., RNA, DNA, PNA) of primer and primerbinding region of the oligonucleotide template, as well as theconcentration of primer and template polynucleotides.

Insofar as it is convenient to use buffer conditions that are compatiblewith DNA polymerase activity and/or ribonuclease activity, stringency ofhybridization of composite primers can be controlled by alteringtemperature of the reaction. Examples of relevant conditions include (inorder of increasing stringency): incubation temperatures ofapproximately 15° C., 20° C., 25° C., 30° C., 37° C., 40° C., 45° C.,50° C., 60° C., or more. Accordingly, in some embodiments, compositeprimer random hybridization occurs at a reduced temperature, for exampleat 25° C.-37° C., followed at incubation at increased temperature(s)suitable for the isothermal amplification phase of the methods (such asabout 50° C.). In some embodiments, temperature is increased at 5° C.increments. In other embodiments, temperature is shifted from low tohigh temperature.

Nucleotide and/or nucleotide analogs, such as deoxyribonucleosidetriphosphates, that can be employed for synthesis of the primerextension products in the methods of the invention are provided in theamount of from preferably about 50 to about 2500 μM, more preferablyabout 100 to about 2000 μM, even more preferably about 200 to about 1700μM, and most preferably about 250 to about 1500 μM. In some embodiments,a nucleotide or nucleotide analog whose presence in the primer extensionstrand enhances displacement of the strand (for example, by causing basepairing that is weaker than conventional AT, CG base pairing) isincluded. Such nucleotide or nucleotide analogs include deoxyinosine andother modified bases, all of which are known in the art.

The oligonucleotide components of the amplification reactions of theinvention are generally in excess of the number of target nucleic acidsequence to be amplified. They can be provided at about or at leastabout any of the following: 10, 10², 10⁴, 10⁶, 10⁸, 10¹⁰, 10¹² times theamount of target nucleic acid. Composite primers can each be provided atabout or at least about any of the following concentrations: 50 nM, 100nM, 500 nM, 1 uM, 2.5 uM, 5 uM, 10 uM. Composite primer concentrationalso impacts frequency and/or position of composite primerhybridization. Generally, increased primer concentrations increasedfrequency of primer hybridization. Auxiliary primers can be provided atabout or at least about any of the following concentrations: about 25nM, about 50 nM, about 100 nM, about 500 nM, about 1 uM, about 2.5 uM,about 5 uM, about 10 uM, or more.

In one embodiment, the foregoing components are added simultaneously atthe initiation of the amplification process. In another embodiment,components are added in any order prior to or after appropriatetimepoints during the amplification process, as required and/orpermitted by the amplification reaction. Such timepoints, some of whichare noted below, can be readily identified by a person of skill in theart. The enzymes used for nucleic acid amplification according to themethods of the invention can be added to the reaction mixture eitherprior to the target nucleic acid denaturation step, following thedenaturation step, or following hybridization of the primer to thetarget polynucleotide, as determined by their thermal stability and/orother considerations known to the person of skill in the art. In theseembodiments, the reaction conditions and components may be variedbetween the different reactions.

The amplification process can be stopped at various timepoints, andresumed at a later time. Said timepoints can be readily identified by aperson of skill in the art. One timepoint is at the end of randomcomposite primer hybridization. Another timepoint is at the end ofrandom composite primer hybridization and composite primer extensionproduct synthesis. Another timepoint (in some embodiments) is followingcleavage of template RNA. Another timepoint is immediately prior toinitiation of single primer isothermal amplification (which in someembodiments, may be initiated by addition of an enzyme (such as RNase H)that cleaves RNA from RNA/DNA heteroduplex, and optionally, DNApolymerase). Another timepoint is at the end of second primer extensionproduct synthesis. Methods for stopping the reactions are known in theart, including, for example, cooling the reaction mixture to atemperature that inhibits enzyme activity or heating the reactionmixture to a temperature that destroys an enzyme. Methods for resumingthe reactions are also known in the art, including, for example, raisingthe temperature of the reaction mixture to a temperature that permitsenzyme activity, replenishing a destroyed (depleted) enzyme, or addingreagent(s) necessary for initiation of a step (for example, addition ofRNase H and/or DNA polymerase to initiate the single primer isothermalamplification phase of the methods). In some embodiments, one or more ofthe components of the reactions is replenished prior to, at, orfollowing the resumption of the reactions. For example, it may benecessary to replenish the composite primer prior to beginning thesingle primer isothermal amplification reaction if the same compositeprimer is being used. Alternatively, the reaction can be allowed toproceed (i.e., from start to finish) without interruption.

The reaction can be allowed to proceed without purification ofintermediate complexes, for example, to remove primer. Products can bepurified at various timepoints, which can be readily identified by aperson of skill in the art. One timepoint is at the end of formation ofthe complex comprising an RNA/DNA partial heteroduplex. Anothertimepoint is at the end of random composite primer hybridization.

The detection of the amplification product is indicative of the presenceof the target sequence. Quantitative analysis is also feasible. Directand indirect detection methods (including quantitation) are well knownin the art. For example, by comparing the amount of product amplifiedfrom a test sample containing an unknown amount of a polynucleotidecontaining a target sequence to the product of amplification of areference sample that has a known quantity of a polynucleotide thatcontains the target sequence, the amount of target sequence in the testsample can be determined.

Compositions and Kits of the Invention

The invention also provides compositions and kits used in the methodsdescribed herein. The compositions may be any component(s), reactionmixture and/or intermediate described herein, as well as anycombination.

In one embodiment, the invention provides a composition comprising acomposite primer as described herein. In some embodiments, the compositeprimer comprises an RNA portion adjacent to the DNA portion. In anotherembodiment, the composite primer comprises 5′- and 3′-DNA portions withat least one intervening RNA portion. In other embodiments, the RNAportion of the composite primer consists of 7 to about 20 nucleotidesand the DNA portion of the composite primer consists of about 5 to about20 nucleotides. In still other embodiments, the RNA portion of thecomposite primer consists of about 10 to about 20 nucleotides and theDNA portion of the composite primer consists of about 7 to about 20nucleotides. In some embodiments, the composite primer is selected fromthe following composite primers: 5′-GACGGAUGCGGUCUdCdCdAdGdTdGdT-3 (SEQID NO:1); and 5′-CGUAUUCUGACGACGUACUCdTdCdAdGdCdCdT-3′ (SEQ ID NO:2),wherein italics denote ribonucleotides and “d” denotesdeoxyribonucleotides.

In other examples, the invention provides a composition comprising acomposite primer as described herein, and auxiliary primers (forexample, a population of randomized hexamer primers). In someembodiments, the composite primer is selected from the followingcomposite primers: 5′-GACGGAUGCGGUCUdCdCdAdGdTdGdT-3 (SEQ ID NO:1); and5′-CGUAUUCUGACGACGUACUCdTdCdAdGdCdCdT-3′ (SEQ ID NO:2), wherein italicsdenote ribonucleotides and “d” denotes deoxyribonucleotides.

In other examples, the invention provides a composition comprising acomposite primer that is derivatized by attachment of a moiety capableof effecting attachment of a polynucleotide comprising the compositeprimer to a solid substrate used in preparing nucleic acid microarrays.In some embodiments, the composite primer is further by attachment of apositively charged moiety such as an amine. In other embodiments, thecomposite primer is labeled, for example by derivatizing the compositeprimer with a detectable moiety, such as a label, or a moiety that canbe covalently or non-covalently attached to a label. Labeled compositeprimers are further described herein.

In other examples, the invention provides composition comprising acomposite primer and one or more of: a DNA polymerase; an enzyme thatcleaves RNA from an RNA/DNA duplex; and auxiliary primers (for example,a population of random hexamer primers). In some embodiments, thecomposition further comprises a labeled dNTP. In still otherembodiments, the composition comprises a non-canonical nucleotide (suchas dUTP), and reagents suitable for labeling and/or fragmenting abasicsites, as described in U.S. Patent Application Publication No.2004/0005614 and Kurn et al, co-pending U.S. patent application No.60/533,381.

The compositions are generally in lyophilized or aqueous form,preferably in a suitable buffer.

The invention also provides compositions comprising the amplificationproducts described herein. Accordingly, the invention provides apopulation of DNA which are copies or the complement of a targetsequence, which are produced by any of the methods described herein (orcompositions comprising the products). The invention also includescompositions and various configurations (such as arrays) of thesepopulations, which may be homogeneous (same sequence) or heterogeneous(different sequence). These populations may be any assembly of sequencesobtained from the methods described herein.

The compositions are generally in a suitable medium, although they canbe in lyophilized form. Suitable media include, but are not limited to,aqueous media (such as pure water or buffers).

The invention provides kits for carrying out the methods of theinvention. Accordingly, a variety of kits are provided in suitablepackaging. The kits may be used for any one or more of the usesdescribed herein, and, accordingly, may contain instructions for any oneor more of the following uses: methods of amplification; genotyping,nucleic acid mutation detection (including methods of genotyping),determining the presence or absence of a sequence of interest,quantitating a sequence of interest, preparation of an immobilizednucleic acid (which can be a nucleic acid immobilized on a microarray),comparative genomic hybridization, and characterizing nucleic acidsusing the amplified nucleic acid products generated by the methods ofthe invention, methods of expression profiling, subtractivehybridization and the preparation of probes for subtractivehybridization, and methods of preparing libraries (which may be cDNAand/or differential hybridization libraries).

The kits of the invention comprise one or more containers comprising anycombination of the components described herein, and the following areexamples of such kits. A kit may comprise any of the composite primersdescribed herein. In some embodiments, the kit comprises one or morecomposite primer selected from the following composite primers:5′-GACGGAUGCGGUCUdCdCdAdGdTdGdT-3 (SEQ ID NO:1); and5′-CGUAUUCUGACGACGUACUCdTdCdAdGdCdCdT-3′ (SEQ ID NO:2), wherein italicsdenote ribonucleotides and “d” denotes deoxyribonucleotides. In someembodiments, a kit further comprises auxiliary primers, which may or maynot be separately packaged. The composite primer may be labeled orunlabeled. Kits may also optionally further include any of one or moreof the enzymes described herein (for example, DNA-dependent DNApolymerase, RNA-dependent DNA polymerase, a DNA polymerase that providesboth DNA-dependent and RNA-dependent DNA polymerase activities, and anenzyme capable of cleaving RNA from an RNA/DNA hybrid, such as RNase H),as well as deoxynucleoside triphosphates (labeled or unlabeled orderivatized). Kits may also include one or more suitable buffers (forexample, as described herein). Kits may also include a labeled dNTP(s)and/or a non-canonical nucleotide (such as dUTP), as described in Kurnet al, co-pending U.S. patent application No. 60/381,457.

One or more reagents in the kit can be provided as a dry powder, usuallylyophilized, including excipients, which on dissolution will provide fora reagent solution having the appropriate concentrations for performingany of the methods described herein. Each component can be packaged inseparate containers or some components can be combined in one containerwhere cross-reactivity and shelf life permit.

The kits of the invention may optionally include a set of instructions,generally written instructions, although electronic storage media (e.g.,magnetic diskette or optical disk) containing instructions are alsoacceptable, relating to the use of components of the methods of theinvention for the intended nucleic acid amplification, and/or, asappropriate, for using the amplification products for purposes such asdetection of sequence mutation. The instructions included with the kitgenerally include information as to reagents (whether included or not inthe kit) necessary for practicing the methods of the invention,instructions on how to use the kit, and/or appropriate reactionconditions.

In another example, the kits of the invention comprise a complex ofcomposite primer extension product and second primer extension product.In yet another example, any of these kits further comprises one or morecontrols (which can be, for example, template polynucleotide (e.g., DNAtemplate such as genomic DNA or RNA template such as total RNA or mRNA),composite primers, and/or auxiliary primer(s).

The component(s) of the kit may be packaged in any convenient,appropriate packaging. The components may be packaged separately, or inone or multiple combinations.

The relative amounts of the various components in the kits can be variedwidely to provide for concentrations of the reagents that substantiallyoptimize the reactions that need to occur to practice the methodsdisclosed herein and/or to further optimize the sensitivity of anyassay.

The invention also provides systems for effecting the methods describedherein. These systems comprise various combinations of the componentsdiscussed above.

Any of the systems embodiments may also comprise a template (target)sequence, as described herein. A system generally includes one or moreapparatuses for performing the amplification methods of the invention.Such apparatuses include, for example, heating devices (such as heatingblocks or water baths) and apparatuses which effect automation of one ormore steps of the methods described herein. The methods of the inventionare particularly suitable for use with miniaturized devices, as thermalcycling is not required for any of the steps. A non-limiting example ofsuitable devices includes the BioAnalyzer (Agilant and Caliper) and theeSensor.

The invention also provides reaction mixtures (or compositionscomprising reaction mixtures) which contain various combinations ofcomponents described herein. Examples of reaction mixtures have beendescribed. In some embodiments, the invention provides reaction mixturescomprising (a) a target polynucleotide; (b) a composite primercomprising a 3′ DNA portion and an RNA portion; (c) auxiliary primers;and (d) DNA polymerase. As described herein, any of the compositeprimers may be in the reaction mixture (or a plurality of compositeprimers), including a composite primer that comprises a 5′ RNA portionwhich is adjacent to the 3′ DNA portion. The reaction mixture could alsofurther comprise an enzyme which cleaves RNA from an RNA/DNA hybrid,such as RNase H. In some embodiments, the composite primer is selectedfrom the following composite primers: 5′-GACGGAUGCGGUCUdCdCdAdGdTdGdT-3(SEQ ID NO:1); and 5′-CGUAUUCUGACGACGUACUCdTdCdAdGdCdCdT-3′ (SEQ IDNO:2), wherein italics denote ribonucleotides and “d” denotesdeoxyribonucleotides.

Other reaction mixtures are described herein and are encompassed by theinvention.

The invention also includes compositions comprising any of the complexes(which are intermediates in the methods described herein) describedherein. Examples of such complexes are schematically depicted in FIGS.1-8. As an example, one complex of the invention is a complexcomprising: (a) a target polynucleotide strand; and (b) a compositeprimer, said composite primer comprising a 3′ DNA portion and an RNAportion. The composite primer may have an RNA portion which is 5′ andadjacent to the 3″ DNA portion. As another example, a complex of theinvention is a complex comprising: (a) a composite primer extensionproduct; and (b) a target polynucleotide.

In yet another example, a complex of the invention is a complexcomprising a RNA/DNA partial heteroduplex, prepared by any of themethods described herein. In some embodiments, the complex furthercomprises a second RNA/DNA partial heteroduplex at a second end. In yetanother example, the complex of the invention is a complex comprising a3′ single stranded DNA portion produced by any of the methods describedherein. In some embodiments, the complex further comprises a second 3′single stranded region. In another example, the complex of the inventionis (a) a complex comprising a 3′ single stranded DNA portion, and (b) acomposite primer hybridized to the 3′ single stranded portion.

Methods Using the Amplification Methods and Compositions of theInvention

The methods and compositions of the invention can be used for a varietyof purposes. For purposes of illustration, methods of nucleic acidmutation detection (including methods of genotyping), determining thepresence or absence of a sequence of interest, quantitating a sequenceof interest, preparation of an immobilized nucleic acid (which can be anucleic acid immobilized on a microarray), comparative genomichybridization, and characterizing nucleic acids using the amplifiednucleic acid products generated by the methods of the invention,detecting and/or identifying novel nucleic acid sequences (such as novelcoding or non-coding transcripts), and characterization of splicevariant sequences, are described. Methods of expression profiling,methods of subtractive hybridization and the preparation of probes forsubtractive hybridization, and methods of preparing libraries (which canbe cDNA and/or differential hybridization libraries) are also described.

Method of Preparing Nucleic Acids Immoblized to a Substrate, Including aMicroarray of Nucleic Acids

The products of some of the amplification methods of the invention aresuitable for immobilizing to a surface. In so far as the amplificationproducts of the methods of the invention generally comprises a mixtureof sequences corresponding to sense and antisense copies of templatepolynucleotide, it is useful to immobilize a population of sequencesgenerated by amplification of template polynucleotide from a definedsource (e.g., DNA or RNA from a defined cell population or a singlecell; organism-specific template (for example, the DNA or RNA ofspecific viruses or other pathogen(s) sufficient to identify theorganism); or a disease-specific template. Immobilized amplificationproduct may then be probed with different probes and the hybridizationsignals can be compared. For example, an immobilized array of genomicpolynucleotides (DNA or RNA) from a known pathogen or non-pathogen(suchas a virus, or group of viruses) may be used for assessment of thepresence or identity of a pathogen within a sample of genetic material.Such arrays would be of use in disease surveillance and inidentification of a pathogenic agent in the event of a disease outbreak.Polynucleotides may be isolated from a suspected sample, labeled usingany method known in the art, and hybridized to such an array. Thedetection of signal due to hybridization to the array providesinformation as to the presence or identity of a pathogen present insample polynucleotide.

Amplification products can be attached to a solid or semi-solid supportor surface, which may be made, e.g., from glass, plastic (e.g.,polystyrene, polypropylene, nylon), polyacrylamide, nitrocellulose, orother materials.

Several techniques are well-known in the art for attaching nucleic acidsto a solid substrate such as a glass slide. One method is to incorporatemodified bases or analogs that contain a moiety that is capable ofattachment to a solid substrate, such as an amine group, a derivative ofan amine group or another group with a positive charge, into theamplified nucleic acids. The amplification product is then contactedwith a solid substrate, such as a glass slide, which is coated with analdehyde or another reactive group which will form a covalent link withthe reactive group that is on the amplification product and becomecovalently attached to the glass slide. Microarrays comprising theamplification products can be fabricated using a Biodot (BioDot, Inc.Irvine, Calif.) spotting apparatus and aldehyde-coated glass slides (CELAssociates, Houston, Tex.). Amplification products can be spotted ontothe aldehyde-coated slides, and processed according to publishedprocedures (Schena et al., Proc. Natl. Acad. Sci. U.S.A. (1995)93:10614-10619). Arrays can also be printed by robotics onto glass,nylon (Ramsay, G., Nature Biotechnol. (1998), 16:40-44), polypropylene(Matson, et al., Anal Biochem. (1995), 224(1):110-6), and siliconeslides (Marshall, A. and Hodgson, J., Nature Biotechnol. (1998),16:27-31). Other approaches to array assembly include finemicropipetting within electric fields (Marshall and Hodgson, supra), andspotting the polynucleotides directly onto positively coated plates.Methods such as those using amino propyl silicon surface chemistry arealso known in the art, as disclosed at http://www.cmt.corning.com andhttp://cmgm.stanford.edu/pbrown/.

One method for making microarrays is by making high-densitypolynucleotide arrays. Techniques are known for rapid deposition ofpolynucleotides (Blanchard et al., Biosensors & Bioelectronics,11:687-690). Other methods for making microarrays, e.g., by masking(Maskos and Southern, Nuc. Acids. Res. (1992), 20:1679-1684), may alsobe used. In principle, and as noted above, any type of array, forexample, dot blots on a nylon hybridization membrane, could be used.However, as will be recognized by those skilled in the art, very smallarrays will frequently be preferred because hybridization volumes willbe smaller.

The amplified polynucleotides may be spotted as a matrix on substratescomprising paper, glass, plastic, polystyrene, polypropylene, nylon,polyacrylamide, nitrocellulose, silicon, optical fiber or any othersuitable solid or semi-solid (e.g., thin layer of polyacrylamide gel(Khrapko, et al., DNA Sequence (1991), 1:375-388) surface.

An array may be assembled as a two-dimensional matrix on a planarsubstrate or may have a three-dimensional configuration comprising pins,rods, fibers, tapes, threads, beads, particles, microtiter wells,capillaries, cylinders and any other arrangement suitable forhybridization and detection of target molecules. In one embodiment thesubstrate to which the amplification products are attached is magneticbeads or particles. In another embodiment, the solid substrate comprisesan optical fiber. In yet another embodiment, the amplification productsare dispersed in fluid phase within a capillary which, in turn, isimmobilized with respect to a solid phase.

Arrays may also be composed of particles, such as beads. The beads maybe labeled with the amplified products alone, or may be labeled withboth the amplified products and an additional label, such as defineddyes or other labels.

Characterization of Nucleic Acids

The amplification products obtained by the methods of the invention areamenable to further characterization. The products of the methods of theinvention are particularly amenable to quantitative analysis, assufficient DNA is produced which generally accurately reflect therepresentation of the various polynucleotides in the starting material.

The amplified polynucleotide products (i.e., products of any of theamplification methods described herein), can be analyzed using, forexample, probe hybridization techniques known in the art, such asSouthern and Northern blotting, and hybridizing to probe arrays. Theycan also be analyzed by electrophoresis-based methods, such asdifferential display and size characterization, which are known in theart. In addition, the polynucleotide products may serve as startingmaterial for other analytical and/or quantification methods known in theart, such as real time PCR, quantitative TaqMan, quantitative PCR usingmolecular beacons, methods described in U.S. Pat. Nos. 6,251,639,6,686,156, and 6,692,918; U.S. Patent Publication Nos. 2002/0115088 A1,2003/0186234 A1, 2003/0087251A1, 2002/0164628, and 2003/0215926, andInternational Patent Application Publication WO 03/08343. Thus, theinvention includes those further analytical and/or quantificationmethods as applied to any of the products of the methods herein.

In one embodiment, the amplification methods of the invention areutilized to generate multiple copies of polynucleotide products, andproducts are analyzed by contact with a probe.

In some embodiments, the amplification methods of the invention areutilized to generate multiple copies of single stranded polynucleotide(generally, DNA) products that are labeled by using composite primersthat are labeled (in the portion(s) that is not cleaved). For example,the primer can be labeled with an aminoallyl labeled nucleotide. Inother embodiments, the amplification methods of the invention areutilized to generate multiple copies of polynucleotide (generally, DNA)products that are labeled by the incorporation of labeled nucleotidesduring DNA. For example, amplification according to the methods of theinvention can be carried out with suitable labeled dNTPs. These labelednucleotides can be directly attached to a label, or can comprise amoiety which could be attached to a label. The label may be attachedcovalently or non-covalently to the amplification products. Suitablelabels are known in the art, and include, for example, a ligand which isa member of a specific binding pair which can be detected/quantifiedusing a detectable second member of the binding pair. Thus,amplification of template polynucleotide according to the methods of theinvention in the presence of, for example, Cy3-dUTP or Cy5-dUTP resultsin the incorporation of these nucleotides into the amplificationproducts. Amplification can also be in the presence of anaminoallyl-derivatized nucleotide, such as aminoallyl dUTP.Amplification product comprising aminoallyl dUTP can be coupled to alabel, such as Cy3 or Cy5.

In other embodiments, the methods of the amplification are performed inhe present of a non-canonical nucleotide, e.g., dUTP, and amplificationcomprising a non-canonical nucleotide is labeled and/or fragmentedaccording to the methods disclosed in U.S. Patent ApplicationPublication No. 2004/0005614. Briefly, non-canonical nucleotide (whenincorporated into amplification product) is cleaved, generating anabasic site. The abasic site is then labeled by contacting with areagent capable of labeling an abasic site. The polynucleotidecomprising an abasic site can also be cleaved at the abasic site,generating fragments suitable for further analysis, e.g., hybridizationto an array. The fragments can also be labeled as described above.

The labeled amplification products are particularly suitable foranalysis (for example, detection and/or quantification) by contactingthem with, for example, microarrays (of any suitable surface, whichincludes glass, chips, plastic), beads, or particles, that comprisesuitable probes such as cDNA and/or oligonucleotide probes. Thus, theinvention provides methods to characterize (for example, detect and/orquantify) an target polynucleotide of interest by generating labeledpolynucleotide (generally, DNA) products using amplification methods ofthe invention, and analyzing the labeled products. Analysis of labeledproducts can be performed by, for example, hybridization of the labeledamplification products to, for example, probes immobilized at, forexample, specific locations on a solid or semi-solid substrate, probesimmobilized on defined particles, or probes immobilized on blots (suchas a membrane), for example arrays, which have been described above.Other methods of analyzing labeled products are known in the art, suchas, for example, by contacting them with a solution comprising probes,followed by extraction of complexes comprising the labeled amplificationproducts and probes from solution. The identity of the probes providescharacterization of the sequence identity of the amplification products,and thus by extrapolation the identity of the target polynucleotidepresent in a sample. Hybridization of the labeled products isdetectable, and the amount of specific labels that are detected isproportional to the amount of the labeled amplification products of aspecific target polynucleotide of interest.

The amount of labeled products (as indicated by, for example, detectablesignal associated with the label) hybridized at defined locations on anarray can be indicative of the detection and/or quantification of thecorresponding target polynucleotide species in the sample.

Methods of characterization include sequencing by hybridization (see,e.g., Dramanac, U.S. Pat. No. 6,270,961) and global genomichybridization (also termed comparative genome hybridization) (see, e.g.,Pinkel, U.S. Pat. No. 6,159,685; Daigo et al (2001) Am. J. Pathol. 158(5):1623-1631. Briefly, comparative genome hybridization comprisespreparing a first population of labeled polynucleotides (which can bepolynucleotide fragments) according to any of the methods describedherein, wherein the template from which the first population issynthesized is total genomic DNA. A second population of labeledpolynucleotides (to which the first population is desired to becompared) is prepared from a second genomic DNA template. The first andsecond populations are labeled with different labels. The hybridizedfirst and second populations are mixed, and hybridized to an array orchromosomal spread. The different labels are detected and compared.

In another aspect, the invention provides a method of quantitatinglabeled and/or fragmented nucleic acids comprising use of anoligonucleotide (probe) of defined sequence (which may be immobilized,for example, on a microarray).

The amplification products generated according to the methods of theinvention are also suitable for analysis for the detection of anyalteration in the target nucleic acid sequence, as compared to areference nucleic acid sequence which is identical to the target nucleicacid sequence other than the sequence alteration. When the targetpolynucleotide is genomic DNA or RNA, the sequence alterations may besequence alterations present in the genomic sequence or may be sequencealterations which are not reflected in the genomic sequence, forexample, alterations due to post transcriptional alterations, and/ormRNA processing, including splice variants. Sequence alterations(interchangeably called “mutations”) include deletion, substitution,insertion and/or transversion of one or more nucleotide.

Other art recognized methods of analysis for the detection of anyalteration in the target nucleic acid sequence, as compared to areference nucleic acid sequence, are suitable for use with the nucleicacid products of the amplification methods of the invention. Suchmethods are well-known in the art, and include various methods for thedetection of specific defined sequences including methods based onallele specific primer extension, allele specific probe ligation,differential probe hybridization, and limited primer extension. See, forexample, Kurn et al, U.S. Pat. No. 6,251,639 B1; U.S. Pat. Nos.5,888,819; 6,004,744; 5,882,867; 5,854,033; 5,710,028; 6,027,889;6,004,745; 5,763,178; 5,011,769; 5,185,243; 4,876,187; 5,882,867;5,731,146; WO US88/02746; WO 99/55912; WO 92/15712; WO 00/09745; WO97/32040; WO 00/56925; and U.S. Pat. No. 5,660,988. Thus, the inventionalso provides methods for detection of a mutation in a targetpolynucleotide comprising a mutation (which can be a single nucleotidepolymorphism), comprising: (a) amplifying a target polynucleotide usingany of the methods described herein; and (b) analyzing the amplificationproducts for presence of an alteration (mutation) as compared to areference polynucleotide.

It is understood that the amplification products can also serve astemplate for further analysis such as sequence, polymorphism detection(including multiplex SNP detection) using, e.g., oligonucleotideligation-based assays, analysis using Invader, Cleavase or limitedprimer extension, and the like. For methods that generally requirelarger volumes of input material, the methods of the invention may beused to “pre” amplify a pool of polynucleotides to generate sufficientinput material for subsequent analysis.

Determination of Gene Expression Profile

The amplification methods of the invention are particularly suitable foruse in determining the levels of expression of one or more genes in asample since the methods described herein are capable of amplifying amultiplicity, including a large multiplicity of target RNAs in the samesample. As described above, amplification products can be detected andquantified by various methods, as described herein and/or known in theart. Since RNA is a product of gene expression, the levels of thevarious RNA species, such as mRNAs, in a sample is indicative of therelative expression levels of the various genes (gene expressionprofile). Thus, determination of the amount of RNA sequences of interestpresent in a sample, as determined by quantifying amplification productsof the sequences, provides for determination of the gene expressionprofile of the sample source.

Accordingly, the invention provides methods of determining geneexpression profile in a sample, said method comprising: amplifyingsingle stranded product from template RNA s in the sample, using any ofthe methods described herein; and determining amount of amplificationproducts of each RNA, wherein each said amount is indicative of amountof each RNA in the sample, whereby the expression profile in the sampleis determined. Generally, labeled products are generated. In certainembodiments, the target RNA is mRNA. It is understood that amount ofamplification product may be determined using quantitative and/orqualitative methods. Determining amount of amplification productincludes determining whether amplification product is present or absent.Thus, an expression profile can includes information about presence orabsence of one or more RNA sequence of interest. “Absent” or “absence”of product, and “lack of detection of product” as used herein includesinsignificant, or de minimus levels.

The methods of expression profiling are useful in a wide variety ofmolecular diagnostic, and especially in the study of gene expression inessentially any mammalian cell (including a single cell) or cellpopulation. A cell or cell population (e.g. a tissue) may be from, forexample, blood, brain, spleen, bone, heart, vascular, lung, kidney,pituitary, endocrine gland, embryonic cells, tumors, or the like.Expression profiling is also useful for comparing a control (normal)sample to a test sample, including test samples collected at differenttimes, including before, after, and/or during development, a treatment,and the like.

Method of Preparing a Library

The DNA products of the methods of the invention are useful in preparinglibraries, including cDNA libraries and subtractive hybridizationlibraries. Using the methods of the invention, libraries may be preparedfrom limited amount of starting material, for example, mRNA extractedfrom limited amount of tissue or even single cells. Accordingly, in oneaspect, the methods of the invention provides preparing a library fromthe DNA products of the invention. In still another aspect, theinvention provides methods for making a library, said method comprising:preparing a subtractive hybridization probe using any of the methodsdescribed herein.

Methods of Subtractive Hybridization

The amplification methods of the invention are particularly suitable foruse in subtractive hybridization methods, in which (at least) a firstand second target polynucleotide population is compared, since themethods described herein are capable of amplifying multiple targetpolynucleotides in the same sample, and the methods of the invention aresuitable for producing large amounts of single stranded antisensenucleic acid suitable for use as “driver” in subtractive hybridization.For example, two nucleic acid populations, one sense and one antisense,can be allowed to mix together with one population present in molarexcess (“driver”). Sequence present in both populations will formhybrids, while sequences present in only one population remainsingle-stranded. Thereafter, various well known techniques are used toseparate the unhybridized molecules representing differentiallyexpressed sequences. See, e.g., Hamson et al., U.S. Pat. No. 5,589,339;Van Gelder, U.S. Pat. No. 6,291,170. The methods of subtractivehybridization provided herein are particularly suited for subtractivehybridization using amplified target RNAs.

Accordingly, the invention provides methods for performing subtractivehybridization, said methods comprising: (a) preparing multiple DNAcopies of the complement of target polynucleotide from a firstpolynucleotide population using any of the amplification methodsdescribed herein; and (b) hybridizing the multiple copies to a secondpolynucleotide population, whereby a subpopulation of the secondpolynucleotide population forms a complex with DNA copies of the firstpolynucleotide population. The invention also provides methods forperforming subtractive hybridization, said methods comprising:hybridizing multiple copies of the complement of at least onepolynucleotide from a first polynucleotide population using any of theamplification methods described herein to a second polynucleotidepopulation, whereby a subpopulation of the second population forms acomplex with a copy from the copies of the first polynucleotidepopulation. In preferred embodiments, the polynucleotide populationsutilized in subtractive hybridization are RNA populations. In someembodiments, “driver” single stranded anti-sense DNA product of themethods of the invention is combined with tester (sense) RNA species. Insome embodiments, “driver” single stranded antisense nucleic acid(generally, DNA) product is produced using the methods of the inventiondescribed herein.

In another aspect, the invention provides methods of differentialamplification in which single stranded driver (antisense) DNA sequencesthat hybridize with tester RNA sequence are subjected to cleavage by anagent that cleaves RNA present in a DNA/RNA hybrid, such as RNase H.Cleavage of the RNA results in the inability to generate single strandedDNA product from the test RNA strands. Conversely, non-cleaved tester(i.e., tester RNA that did not hybridize to driver DNA molecules) mayserve as a substrate for subsequent amplification. Amplifieddifferentially expressed products have many uses, including as adifferential expression probe, to produce differential expressionlibraries. Accordingly, the invention provides methods for differentialamplification of one or more RNA template sequence, said methodcomprising: (a) preparing multiple polynucleotide (generally, DNA)copies of the complement of RNA from a first RNA population using any ofthe amplification methods described herein; (b) hybridizing the multiplecopies to a second RNA population, whereby a subpopulation of the secondRNA population forms a complex with a DNA copy; (c) cleaving RNA in thecomplex of step (b) with an enzyme that cleaves RNA from an RNA/DNAhybrid; and (d) amplifying an unhybridized subpopulation of the secondRNA population, whereby multiple copies of single stranded DNAcomplementary to the unhybridized subpopulation of the second RNApopulation are generated. In some embodiments, step (d) is performedusing any of the amplification methods described herein. In someembodiments, the methods comprise hybridizing multiple polynucleotide(generally, DNA) copies of the complement of at least one RNA sequencesof interest from a first RNA population using any of the amplificationmethods described herein to a second RNA population, whereby asubpopulation of the second RNA population forms a complex with a DNAcopy; (b) cleaving RNA in the complex of step (a) with an enzyme thatcleaves RNA from an RNA/DNA hybrid; and (c) amplifying an unhybridizedsubpopulation of the second RNA population, whereby multiple copies ofsingle stranded DNA complementary to the unhybridized subpopulation ofthe second RNA population are generated.

The following Examples are provided to illustrate, but not limit, theinvention.

EXAMPLES Example 1 Global Amplification of Human Genomic DNA Using aComposite Primer and Random Hexamer Primers

Global amplification reactions were performed using composite primerIA20 and human genomic DNA as a template. The sequence of compositeprimer IA20 is as follows: IA20: 5′-GACGGAUGCGGUCUdCdCdAdGdTdGdT-3′ (SEQID NO:1), where italics denote ribonucleotides, and “d” denotesdeoxyribonucleotides.

Human genomic DNA (Clontech, Cat. No. 6550-1) was diluted in TE bufferand denatured by heating to 99° C. In some samples, DNA and primers weremixed in amplification buffer and heated at 96° C. for 2-4 minutes.

The following reaction mixture was used.

2 ng of pre-denatured human genomic DNA (approximately 600 copies)

2 μl of random hexamer N6 (final concentration 2.5 μM) (Qiagen-Operon;item No.: PolyN (6-mer));

0.5 μl (100 μM) composite primer IA20 (final concentration: 2.5 μM),

0.1 μl Bst DNA polymerase (0.04 U/μl) (New England BioLabs, Catalog No.M0275);

10 μl buffer (final concentration: 20 mM Tris-HCl, pH 8.5; 5 mM MgCl₂;RNasin, 0.3 U/μl; DTT, 0.5 mM; acetylated BSA, 0.1 μg/μl; T4 gp32protein, 0.15 μg/μl)), and

RNase-free water to final volume of 15 μl.

The mixture was incubated at 30° C. for 5 minutes, followed by 5 minutesat 40° C., and 2 minutes at 50° C.

5 μl of enzyme mixture (RNase H, final concentration of 0.025 U/μl; andBst DNA polymerase (large fragment), final concentration 0.2 U/μl) wasadded, and the reaction was incubated at 50° C. for 30-40 minutes. Thereaction was stopped by incubation at 80° C. for 5 minutes to inactivatethe enzymes.

Control reactions were prepared in which either composite primer, N6random primers, or RNase H were omitted. In control reactions, thecorresponding volume (of the omitted reagent) was replaced by water.

Amplification reaction product was analyzed as follows. 0.5 μl ofreaction mixture was loaded onto 4-20% gradient acrylamide gel andelectrophoresed at 200-220 V constant voltage for 30 min. The gel wasstained in 0.005 mg/ml Ethidium Bromide for 2 minutes and washed inwater for 1 minute. The gel was then visualized and photographed on theAlphaImager 2200 system. The results are shown in FIG. 9. Lanescorrespond to the reaction mixtures containing the following components:

Lanes #1-2: Complete reaction mixture

Lanes #3-4: Reaction lacking N6 random primer.

Lanes #5-6: Reaction lacking composite primer.

Lanes #7-8: Reaction lacking RNase H.

FIG. 9 shows that amplification product of varying molecular weight wasproduced in reactions containing the complete reaction mixture(described above), and in reaction lacking N6 random primers. However,reactions in which composite primer or RNase H were omitted did not showdetectable reaction product.

Amplification product was quantified using the following procedure:reactions were prepared and processed as described above. Reactionmixture was diluted 100-fold and 2 μl of the diluted samples were usedin Real Time PCR quantification with the following primer pairs thatamplify a single copy sequence on Chromosome 7:

221PF2 (5′-AGTATCTGGCACATCTT-3′ (SEQ ID NO:_(———))) and 221PR2(5′-GGGAGATATTATTTGGC-3′ (SEQ ID NO:_(———))).

Amplification with primers 221PF2 and 221 PR2 was expected to yield a 62base pair PCR product. The PCR reaction mixture contained: 1 μl of 10 μMof each primer, 6 μl of water, 10 μl of 2×SYBR Green PCR Master Mix(Applied Biosystems), and 2 μl diluted reaction mixture (diluted asdescribed above). A control reaction was conducted using 2 μl humangenomic DNA, instead of amplification reaction product as a template.

The thermal cycling program used was: one cycle of 94° C. for 10 min.,followed by 45 cycles of 94° C. for 30 sec., 55° C. for 30 sec., and 72°C. for 30 sec. The Real Time PCR quantification data were presented asCt values (threshold cycle). The values obtained for quantification ofamplification products were compared with that obtained for 2 μl humangenomic DNA (labeled “non-amplified Genomic DNA” in Table 1). Thedilution factor between the diluted global amplification products andthe original human genomic DNA input into the amplification reactions is1000 fold. The data were summarized in Table 1.

TABLE 1 Quantification of amplification products and target humangenomic DNA employing Real Time PCR with SYBR Green. A single copysequence on Chromosome 7 of human genomic DNA (221) was used fordetermination of amplification efficiency. Amplification efficiency isexpressed as the relative amount of this single copy sequence instarting genomic DNA sample and following global amplification. Reactioncomponents Ct Efficiency Complete 29 4000 fold No N6 random primers 36 40 fold No Composite primer None None No RNase H None None None (nonamplified Genomic DNA) 31 None

In a second experiment, human genomic DNA was amplified using compositeprimer IA20 essentially as described above. Following amplification,several target sequences were quantified using Real Time PCR essentiallyas described above. Chromosomal location of target sequences and PCRprimer pairs are shown in Table 2. Table 2 shows the relative amount oftarget sequence following global amplification, and the amplificationefficiency. Real Time PCR quantification data were presented as Ctvalues (threshold cycle).

TABLE 2 Target Real time Amplification location Forward PCR primerReverse PCR primer Delta C(t) fold chromosome #6 GGACGTGTGTTCCTGTTAACACTTTGATCCTGAAAGACT 3.5 2000 (SEQ ID NO_) (SEQ ID NO_) chromosome #7AGTATCTGGCACATCTT GGGAGATATTATTTGGC 4 3000 (SEQ ID NO:_) (SEQ ID NO:_)chromosome #11 AGGTTCCCAGCCTTGGTCC TGAGGCCATGTGTGTGGAAT 2 800 (SEQ IDNO_) (SEQ ID NO:_) chromosome #12 AATAATGTCCAGATATCTTGGTTCCCTACTCCAGCTACTTCT 2.5 1000 (SEQ ID NO:_) (SEQ ID NO:_) chromosome #16CAGCAAGAACACAAGGGAC TCTTGAGAGCGAGGGCA 2.5 1000 (SEQ ID NO_) (SEQ ID NO_)

In a third experiment, human genomic DNA was amplified using compositeprimer BSCA-128F essentially as described above. The sequence ofcomposite primer BSCA-128F is:

5′-CGUAUUCUGACGACGUACUCdTdCdAdGdCdCdT-3′ (SEQ ID NO:2) where italicsdenote ribonucleotides, and “d” denotes deoxyribonucleotides.

Amplification reaction product was analyzed as described above.Amplification product of varying molecular weights was generated,suggesting that the composite primer permitted amplification from amultiplicity of template sequences.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications may be practiced. Therefore, thedescriptions and examples should not be construed as limiting the scopeof the invention.

1. A method for amplification of a template DNA, comprising: (a)incubating a reaction mixture, said reaction mixture comprising: (i) atemplate DNA: (ii) a first primer, wherein the first primer ishybridizable to a multiplicity of template sites, wherein the firstprimer is a population of different composite primers each comprising anRNA portion and a 3′ DNA portion, wherein each composite primercomprises a 3′ random sequence, and wherein each composite primer is atailed primer that comprises a 5′ portion that is not hybridizable tothe template DNA tinder conditions in which the first primer hybridizesto the template DNA; (iii) a DNA-dependent DNA polymerase; and (iv) anRNA-dependent DNA polymerase; wherein the incubation is under conditionsthat permit generation of a complex comprising an RNA/DNA heteroduplex,wherein said complex comprising an RNA/DNA heteroduplex is produced byhybridization of the first primer to the template DNA, primer extensionto generate a first primer extension product, hybridization of a secondprimer to the first primer extension product, and extension of thesecond primer to generate a second primer extension product; and (b)incubating a reaction mixture, said reaction mixture comprising (i) atleast a portion of the reaction products generated according to step(a); (ii) an amplification primer, wherein said amplification primer isa composite primer comprising an RNA portion and a 3′ DNA portion,wherein the amplification primer comprises some of the sequence of thefirst primer, and wherein the first primer and the amplification primerare different primers; (iii) a DNA-dependent DNA polymerase; and (iv) anagent that cleaves RNA from an RNA/DNA heteroduplex; wherein theincubation is under conditions that permit RNA cleavage, primerhybridization, primer extension, and displacement of the first primerextension product when its RNA is cleaved and another amplificationprimer binds to the second primer extension product and is extended,such that primer extension and strand displacement are repeated, wherebymultiple copies of a an amplification product are generated.
 2. Themethod of claim 1, wherein said DNA-dependent DNA polymerase and saidRNA-dependent DNA polymerase of step (a) are the same enzyme.
 3. Themethod of claim 1, wherein said DNA-dependent DNA polymerase and saidRNA-dependent DNA polymerase of step (a) are different enzymes.
 4. Themethod of claim 1, wherein said first primer and said second primer arethe same primer.
 5. The method of claim 1, wherein said first primer andsaid second primer are different primers.
 6. The method of claim 1,wherein step (b) is initiated by the addition of an agent that cleavesRNA front an RNA/DNA heteroduplex to the reaction mixture of step (a).7. The method of claim 1, wherein the agent that cleaves RNA from anRNA/DNA heteroduplex is an enzyme.
 8. The method of claim 7, whereinsaid agent that cleaves RNA from an RNA/DNA heteroduplex is RNase H. 9.The method of claim 1, wherein the RNA portion of the first primer is 5′with respect to the 3′ DNA portion.
 10. The method of claim 9, whereinthe RNA portion of the amplification primer is 5′ with respect to the 3′DNA portion.
 11. The method of claim 1, wherein the RNA portion of theamplification primer is 5′ with respect to the 3′ DNA portion.
 12. Themethod of claim 11, wherein the 5′ RNA portion of the amplificationprimer is adjacent to the 3′ DNA portion.
 13. The method of claim 1,wherein the RNA portion of the first primer consists of about 7 to about50 nucleotides.
 14. The method of claim 13, wherein the DNA portion ofthe first primer consists of about 5 to about 20 nucleotides.
 15. Themethod of claim 1, wherein the RNA portion of the amplification primerconsists of about 7 to about 50 nucleotides.
 16. The method of claim 15,wherein the DNA portion of the amplification primer consists of about 5to about 20 nucleotides.
 17. The method of claim 1, wherein the reactionmixture of step (b) further comprises a non-canonical nucleotide. 18.The method of claim 17, wherein the non-canonical nucleotide is dUTP.19. The method of claim 1, wherein the reaction mixture of step (b)further comprises a labeled nucleotide.
 20. A method for amplificationof a template DNA, comprising: incubating a reaction mixture, saidreaction mixture comprising: (a) a complex comprising a RNA/DNA partialheteroduplex, wherein the complex is generated by incubating a firstreaction mixture, said first reaction mixture comprising: (i) a templateDNA; (ii) a first primer; wherein the first primer is hybridizable to amultiplicity of template sites, wherein the first primer is a populationof different composite primers each comprising an RNA portion and a 3′DNA portion, wherein the composite primer comprises a 3′ randomsequence, and wherein the composite primer is a tailed primer thatcomprises a 5′ portion that is not hybridizable to the template DNAunder conditions in which the first primer hybridizes to the templateDNA; (iii) a DNA-dependent DNA polymerase; and (iv) an RNA-dependent DNApolymerase; wherein the incubation is under conditions that permithybridization of the first primer to the template DNA, primer extensionto generate a first primer extension product, hybridization of a secondprimer to the first primer extension product, and extension of thesecond primer to generate a second primer extension product, whereby acomplex comprising an RNA/DNA partial heteroduplex is generated; (b) anamplification primer, wherein the amplification primer is a compositeprimer comprising an RNA portion and a 3′ DNA portion, wherein theamplification primer comprises some of the sequence of the first primer,and wherein the first primer and the amplification primer are differentprimers; (c) a DNA-dependent DNA polymerase; and (d) an agent thatcleaves RNA from an RNA/DNA heteroduplex; wherein the incubation isunder conditions that permit RNA cleavage, primer hybridization, primerextension, and displacement of the first primer extension product whenits RNA is cleaved and another amplification primer binds to the secondprimer extension product and is extended, such that primer extension andstrand displacement are repeated, whereby multiple copies of anamplification product are generated.
 21. The method of claim 20, whereinthe agent that cleaves RNA from an RNA/DNA heteroduplex is an enzyme.22. The method of claim 21, wherein the enzyme that cleaves RNA from anRNA/DNA heteroduplex is RNase H.
 23. The method of claim 21, whereinsaid DNA-dependent DNA polymerase and said enzyme that cleaves RNA froman RNA/DNA heteroduplex are the same enzyme.
 24. The method of claim 23,wherein said DNA-dependent DNA polymerase and said enzyme that cleavesRNA from an RNA/DNA heteroduplex are different enzymes.
 25. The methodof claim 20, wherein the RNA portion of the amplification primer is 5′with respect to the 3′-DNA portion.
 26. The method of claim 25, whereinthe 5′ RNA portion of the amplification primer is adjacent to the 3′ DNAportion.
 27. The method of claim 20, wherein the RNA portion of theamplification primer consists of about 7 to about 50 nucleotides. 28.The method of claim 27, wherein the DNA portion of the amplificationprimer consists of about 5 to about 20 nucleotides.
 29. The method ofclaim 20, wherein the RNA portion of the amplification primer consistsof about 10 to about 50 nucleotides.
 30. The method of claim 29, whereinthe DNA portion of the amplification primer consists of about 7 to about20 nucleotides.
 31. The method of claim 20, wherein the reaction mixturefurther comprises a non-canonical nucleotide.
 32. The method of claim31, wherein the non-canonical nucleotide is dUTP.
 33. The method ofclaim 20, wherein the reaction mixture further comprises a labelednucleotide.
 34. A method for amplification of a template DNA,comprising: incubating a reaction mixture, said reaction mixturecomprising: (a) a complex of a first primer extension product and asecond primer extension product, wherein the first primer extensionproduct is generated by extension of a first primer hybridized to thetemplate DNA with a DNA polymerase, wherein the first primer is ahybridizable to a multiplicity of template polynucleotide sites, whereinthe first primer is a population of different composite primers eachcomprising an RNA portion and a 3′ DNA portion, wherein each compositeprimer comprises a 3′ random sequence, and wherein each composite primeris a tailed primer that comprises a 5′ portion that is not hybridizableto the template DNA under conditions in which the first primerhybridizes to the template DNA, and wherein the second primer extensionproduct is generated by extension of a second primer hybridized to thefirst primer extension product; (b) an amplification primer, wherein theamplification is a composite primer comprising an RNA portion and a 3′DNA portion, wherein the amplification primer comprises some of thesequence of the first primer, wherein the first primer and theamplification primer are different primers, and wherein theamplification primer is hybridizable to the second primer extensionproduct; (c) a DNA-dependent DNA polymerase; and (d) an agent thatcleaves RNA from an RNA/DNA heteroduplex; wherein the incubation isunder conditions that permit RNA cleavage, primer hybridization, primerextension, and displacement of the first primer extension product fromthe second primer extension product when its RNA is cleaved and anotheramplification primer binds and is extended, such that primer extensionand strand displacement are repeated, whereby multiple copies of anamplification product are generated.
 35. The method of claim 34, whereinsaid agent that cleaves RNA from a RNA/DNA heteroduplex is an enzyme.36. The method of claim 35, wherein the enzyme that cleaves RNA from aRNA/DNA heteroduplex is RNase H.
 37. The method of claim 35, whereinsaid DNA-dependent DNA polymerase and the enzyme that cleaves RNA froman RNA/DNA heteroduplex are the same enzyme.
 38. The method of claim 35,wherein said DNA-dependent DNA polymerase and the enzyme that cleavesRNA from an RNA/DNA heteroduplex are different enzymes.
 39. The methodof claim 34, wherein the RNA portion of the amplification primer is 5′with respect to the 3′ DNA portion.
 40. The method of claim 39, whereinthe 5′ RNA portion of the amplification primer is adjacent to the 3′ DNAportion.
 41. The method of claim 34, wherein the RNA portion of theamplification primer consists of about 7 to about 50 nucleotides. 42.The method of claim 41, wherein the DNA portion of the amplificationprimer consists of about 5 to about 20 nucleotides.
 43. The method ofclaim 34, wherein the RNA portion of the amplification primer consistsof about 10 to about 50 nucleotides.
 44. The method of claim 43, whereinthe DNA portion of the amplification primer consists of about 7 to about20 nucleotides.
 45. The method of claim 34, wherein the reaction mixturefurther comprises a non-canonical nucleotide.
 46. The method of claim45, wherein the non-canonical nucleotide is dUTP.
 47. The method ofclaim 34, wherein the reaction mixture further comprises a labelednucleotide.
 48. A method for amplification of a template DNA,comprising: (a) randomly priming a template DNA with a first primer,wherein said first primer is hybridizable to a multiplicity of templatepolynucleotide sites, wherein the first primer is a population ofdifferent composite primers each comprising an RNA portion and a 3′ DNAportion, and wherein each composite primer is a tailed primer thatcomprises a 5′ portion that is not hybridizable to the template DNAunder conditions in which the first primer hybridizes to the templateDNA; (b) extending said first primer with a DNA polymerase to generate afirst primer extension product; (c) hybridizing a second primer to thefirst primer extension product; (d) extending said second primer with aDNA-dependent DNA polymerase and an RNA-dependent polymerase to generatea second primer extension product, whereby a complex comprising anRNA/DNA heteroduplex is generated; (e) cleaving RNA from the firstprimer with an agent that cleaves RNA from a RNA/DNA heteroduplex; (f)hybridizing an amplification primer to the second primer extensionproduct, wherein said amplification primer is a composite primercomprising a RNA portion and a 3′ DNA portion, wherein the amplificationprimer comprises some of the sequence of the first primer, and whereinthe first primer and the amplification primer are different primers; (g)extending the hybridized amplification primer by strand displacement DNAsynthesis; (h) cleaving RNA from the amplification primer with an agentthat cleaves RNA from a RNA/DNA heteroduplex, such that anotheramplification primer hybridizes and is extended, whereby multiple copiesof an amplification product are generated.
 49. The method of claim 48,wherein the agent that cleaves RNA from an RNA/DNA heteroduplex is anenzyme.
 50. The method of claim 49, wherein the enzyme that cleaves RNAfrom an RNA/DNA heteroduplex is RNase H.
 51. The method of claim 48,wherein the DNA polymerase of step (b) is a DNA-dependent DNApolymerase.
 52. The method of claim 48, wherein the DNA-dependent DNApolymerase and the RNA-dependent DNA polymerase are the same enzyme. 53.The method of claim 48, wherein the DNA-dependent DNA polymerase and theRNA-dependent DNA polymerase are different enzymes.
 54. The method ofclaim 48, wherein the RNA portion of the first primer is 5′ with respectto the 3′ DNA portion.
 55. The method of claim 48, wherein the RNAportion of the amplification primer is 5′ with respect to the 3′ DNAportion.
 56. The method of claim 55, wherein the 5′ RNA portion of theamplification primer is adjacent to the 3′ DNA portion.
 57. The methodof claim 54, wherein the 5′ RNA portion of the first primer is adjacentto the 3′ DNA portion.
 58. The method of claim 48, wherein the RNAportion of the first primer consists of about 7 to about 50 nucleotides.59. The method of claim 58, wherein the DNA portion of the first primerconsists of about 5 to about 20 nucleotides.
 60. The method of claim 48,wherein the RNA portion of the amplification primer consists of about 7to about 50 nucleotides.
 61. The method of claim 60, wherein the DNAportion of the amplification primer consists of about 5 to about 20nucleotides.
 62. The method of claim 48, wherein step (g) is carried outin the presence of a non-canonical nucleotide.
 63. The method of claim62, wherein the non-canonical nucleotide is dUTP.
 64. The method ofclaim 48, wherein step (g) is carried out in the presence of a labelednucleotide.
 65. The method of claim 20, wherein the RNA portion of thefirst primer is 5′ with respect to the 3′-DNA portion.
 66. The method ofclaim 65, wherein the 5′ RNA portion of the first primer is adjacent tothe 3′ DNA portion.
 67. The method of claim 20, wherein said firstprimer and said second primer are the same primer.
 68. The method ofclaim 67, wherein said first primer and said second primer are differentprimers.
 69. The method of claim 34, wherein the RNA portion of thefirst primer is 5′ with respect to the 3′-DNA portion.
 70. The method ofclaim 69, wherein the 5′ RNA portion of the first primer is adjacent tothe 3′ DNA portion.
 71. The method of claim 34, wherein said firstprimer and said second primer are the same primer.
 72. The method ofclaim 71, wherein said first primer and said second primer are differentprimers.
 73. The method of any of claims 1, 20, 34, or 48, wherein saidtemplate DNA is genomic DNA.
 74. The method of claim 73, wherein saidgenomic DNA comprises a multiplicity of genomic DNA templates.